Protein involved in cancer

ABSTRACT

The present invention relates to new uses of a protein (NKCC1) in the diagnosis, screening, treatment and prophylaxis of breast, lung and/or pancreatic cancer. The invention also provides compositions comprising the protein, including vaccines, antibodies that are immunospecific for the protein and agents which interact with or modulate the expression or activity of the protein or which modulate the expression of the nucleic acid which codes for the protein.

The present invention relates to new uses of a protein (NKCC1) in thediagnosis, screening, treatment and prophylaxis of breast, lung and/orpancreatic cancer. The invention also provides compositions comprisingthe protein, including vaccines, antibodies that are immunospecific forthe protein and agents which interact with or modulate the expression oractivity of the protein or which modulate the expression of the nucleicacid which codes for the protein.

The major challenges in breast, lung and/or pancreatic cancer treatmentis to improve early detection rates, to find new non-invasive markersthat can be used to follow disease progression and identify relapse, andto find improved and less toxic therapies, especially for more advanceddisease where 5 year survival is still very poor. There is a great needto identify targets which are more specific to the cancer cells, ideallyones which are expressed on the surface of the tumour cells so that theycan be attacked by promising new approaches like immunotherapeutics andtargeted toxins.

An ideal protein target for cancer immunotherapy should have arestricted expression profile in normal tissues and be over-expressed intumours, such that the immune response will be targeted to tumour cellsand not against other organs. In addition, the protein target should beexposed on the cell surface, where it will be accessible to therapeuticagents. Tumour specific proteins have been identified for a number ofcancer types, by using techniques such as differential screening of cDNA(Hubert, R. S., et al., 1999, Proc. Natl. Acad, Sci. USA 96,14523-14528; Lucas, S., et al., 2000, Int. J. Cancer 87, 55-60), and thepurification of cell-surface proteins that are recognised bytumour-specific antibodies (Catimel, B., et al., 1996, J. Biol. Chem.271, 25664-25670).

Recently, DNA ‘chips’ containing up to 10,000 expressed sequenceelements have been used to characterise tumour cell gene expression(Dhanasekaran, S. M., et al., 2001, Nature 412, 822-826). However, thereare several reasons why the numerous and extensive previoustranscriptomic analysis of cancers, including breast cancer, may nothave revealed all, or even most, tumour associated proteins. Theseinclude; (i) a lack of correlation between transcript anddisease-associated protein levels, particularly common for membraneproteins that often have a long half-life and as such do not have a highmRNA turnover. Therefore, whilst the difference in protein levelsbetween normal and cancerous cells are consistent it is often difficultto associate changes in the mRNA for a given membrane protein with thecancerous state. (ii) Translocation of a protein in the disease staterather than simply differential levels of the transcript, for example,erbB2/HER2-neu, shows much greater plasma-membrane localisation incancer cells than normal breast cells, and the transcription factorsoestrogen receptor and STAT3 translocate to the nucleus to exert theirtumourigenic effects. (iii) Novel/uncharacterised genes are not highlyrepresented within the ‘closed system’ of a cDNA array where there arerestrictions on the number of expressed sequence elements per chip andthe knowledge and availability of DNA clones.

NKCC1 (the sequence of which can be found under the accession numberP55011 in the SwissProt database, (available at http://www.expasy.org/),and which is shown in FIG. 1, SEQ ID NO:1) is a bumetanide sensitivesodium-potassium-chloride (Na—K—Cl) cotransporter (Payne et al, 1995, JBiol Chem, 270(30): pp 17977-17985). Two isoforms of the Na—K—Clcotransporter have been identified; one located on the apical membraneof absorptive epithelia (NKCC2) and one located on the basolateralmembrane of secretory epithelia (NKCC1). The function of the Na—K—Clcotransporters is to provide electroneutral transport of chloride ionsacross epithelia (in a ratio of 1Na: 1K:2Cl), they work in combinationwith the sodium and potassium channels and the sodium pump to cause anet transport of sodium chloride across membranes. Dysregulation of thistransport mechanism can result in diseases such as Cystic Fibrosis andsecretory diarrhoea.

The human NKCC1 protein was identified by the Payne et al in 1995 (seeabove) by the screening of a human colon carcinoma cell line (T84). Itshows significant similarity to the NKCC1 proteins identified fromelasmobranch, mouse, rat, rabbit and flounder.

Both isoforms of the Na—K—Cl cotransporter show a similar structure withlarge N and C terminal domains and 12 transmembrane segments. NKCC1 issignificantly larger than NKCC2 with an extra 80 amino acids at the Nterminus, interestingly it is the N terminus that shows the greatestvariation between species indicating that this section may not bedirectly involved in the ion transportation. A recent study hasindicated that NKCC1 may have 2 splice variants which show differentialexpression across a variety of tissue types indicating that differentialsplicing may play a regulatory role in NKCC1 activity (Vibat, et al.,Anal Biochem 2001;298(2):218-30).

Over-expression of NKCC1 has been reported in asthmatic subjects by geneexpression profiling (Dolganov G M et al, Genome Res (2001); 11(9):1473-83). This data was then confirmed using immunohistochemistry, whichdemonstrated that in asthmatic subjects NKCC1 expression is increasedwith restricted localisation to goblet cells implicating NKCC1 in thepathophysiology of mucus hypersecretion in asthma.

However, no cancer-associated alteration in the expression of the NKCC1protein indicating the usefulness of NKCC1 in the treatment of breast,lung and/or pancreatic cancer has previously been shown.

The present invention is based on the finding that the expression ofNKCC1 protein is increased in breast, lung and/or pancreatic cancer andNKCC1 mRNA shows restricted expression in a few tissues with elevatedexpression in breast, lung and/or pancreatic cancer, suggesting itsutility as a target for breast, lung and/or pancreatic cancer therapyand diagnosis.

Thus, one aspect of the present invention provides a method of screeningfor and/or diagnosis of breast, lung and/or pancreatic cancer in asubject and/or monitoring the effectiveness of breast, lung and/orpancreatic cancer therapy, which method comprises the step of detectingand/or quantifying in a biological sample obtained from said subject, anNKCC1 polypeptide which:

-   -   a) comprises or consists of the amino acid sequence shown in        FIG. 1 (SEQ ID NO:1);    -   b) is a derivative having one or more amino acid substitutions,        modifications, deletions or insertions relative to the amino        acid sequence shown in FIG. 1 (SEQ ID NO:1) which retains the        immunological or biological activity of NKCC1; or    -   c) is a fragment of a polypeptide having the sequence shown in        FIG. 1 (SEQ ID NO: 1), which is at least ten amino acids long        and has at least 70% sequence identity over the length of the        fragment.

The polypeptides described in a) to c) above are hereinafter referred toas “NKCC1 polypeptides”. The term “polypeptides” includes peptides,polypeptides and proteins. These are used interchangeably unlessotherwise specified.

In the context of the present invention, the biological sample can beobtained from any source, such as a serum sample or a tissue sample,e.g. breast, lung, or pancreatic tissue.

A convenient means for detecting/quantifying the NKCC1 polypeptidesinvolves the use of antibodies, therefore, the NKCC1 polypeptides asdefined herein also find use in raising antibodies. Thus, in a furtheraspect, the present invention provides the use of an antibody whichbinds to at least one NKCC1 polypeptide for screening for and/ordiagnosis of breast, lung and/or pancreatic cancer in a subject.Preferably, the antibody is used for detecting and/or quantifying theamount of an NKCC1 polypeptide in a biological sample obtained from saidsubject.

In one embodiment, binding of antibody in tissue sections can be used todetect aberrant polypeptide localisation or an aberrant level ofpolypeptide. In a specific embodiment, an antibody to an NKCC1polypeptide can be used to assay a patient tissue (e.g. a breast, lungand/or pancreatic biopsy) for the level of the polypeptide where anaberrant level of polypeptide is indicative of breast, lung and/orpancreatic cancer. As used herein, an “aberrant level” means a levelthat is increased or decreased compared with the level in a subject freefrom breast, lung and/or pancreatic cancer or a reference level. In aspecific embodiment an antibody to an NKCC1 polypeptide can be used toassay a patient tissue (e.g. a breast, lung and/or pancreatic biopsy)for the level of the polypeptide where an increased level of the NKCC1polypeptide is indicative of breast, lung and/or pancreatic cancer.

Suitable immunoassays include, without limitation, competitive andnon-competitive assay systems using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, precipitin reactions, geldiffusion precipitin reactions, immunodiffusion assays, agglutinationassays, complement-fixation assays, immunoradiometric assays,fluorescent immunoassays and protein A immunoassays.

In another aspect, the invention provides a method of screening forand/or diagnosis of breast, lung and/or pancreatic cancer in a subjectand/or monitoring the effectiveness of breast, lung and/or pancreaticcancer therapy, which method comprises the step of detecting and/orquantifying in a biological sample obtained from said subject, an NKCC1nucleic acid which:

-   -   d) comprises or consists of the DNA sequence shown in FIG. 1        (SEQ ID NO:2) or its RNA equivalent;    -   e) is a sequence which codes for a polypeptide as defined in        a), b) or c);    -   f) is a sequence which is complementary to the sequences of d)        or e);    -   g) is a sequence which codes for the same polypeptide, as the        sequences of d) or e);    -   h) is a sequence which shows substantial identity with any of        those of d), e), f) or g); or    -   i) is a fragment of d), e), f), g) or h), which is at least 8        nucleotides in length.

The term ‘RNA equivalent’ when used above indicates that a given RNAmolecule has a sequence which is complementary to that of a given DNAmolecule, allowing for the fact that in RNA ‘U’ replaces ‘T’ in thegenetic code. The nucleic acid molecule may be in isolated, recombinantor chemically synthetic form.

Unless the context indicates otherwise, the term “NKCC1 nucleic acid”includes those nucleic acid molecules defined in d) to i) above and mayhave one or more of the following characteristics:

-   -   1) they may be DNA or RNA;    -   2) they may be single or double stranded;    -   3) they may be provided in recombinant form, e.g. covalently        linked to a 5′ and/or a 3′ flanking sequence to provide a        molecule which does not occur in nature;    -   4) they may be provided without 5′ and/or 3′ flanking sequences        which normally occur in nature;    -   5) they may be provided in substantially pure form. Thus they        may be provided in a form which is substantially free from        contaminating proteins and/or from other nucleic acids; and    -   6) they may be provided with introns or without introns (e.g. as        cDNA).

In view of the foregoing description the skilled person will appreciatethat utilisation of a large number of nucleic acids is within the scopeof the present invention.

In another aspect, the present invention provides a method for theprophylaxis and/or treatment of breast, lung and/or pancreatic cancer ina subject, which comprises administering to said subject atherapeutically effective amount of an NKCC1 polypeptide or an NKCC1nucleic acid.

In a yet another aspect, the present invention provides the use of anNKCC1 polypeptide or an NKCC1 nucleic acid in the preparation of acomposition for use in the prophylaxis and/or treatment of breast, lungand/or pancreatic cancer. The subject may be a mammal and is preferablya human.

In the aspects supra, the NKCC1 polypeptides may be provided in isolatedor recombinant form, and may be fused to other moieties. The NKCC1polypeptides thereof may be provided in substantially pure form, that isto say free, to a substantial extent, from other proteins. Thus, anNKCC1 polypeptide may be provided in a composition in which it is thepredominant component present (i.e. it is present at a level of at least50%; preferably at least 75%, at least 80%, at least 85%, at least 90%,at least 95% or at least 98%; when determined on a weight/weight basisexcluding solvents or carriers).

In order to more fully appreciate the present invention, polypeptideswithin the scope of a)-c) above will now be discussed in greater detail.

Polypeptides within the Scope of a)

A polypeptide within the scope of a), may consist of the particularamino acid sequence given in FIG. 1 (SEQ ID NO: 1) or may have anadditional N-terminal and/or an additional C-terminal amino acidsequence relative to the sequence given in FIG. 1 (SEQ ID NO: 1).Additionally, NKCC1 polypeptides may be in the form of a “mature”protein or may be part of a larger protein such as a fusion protein.

Additional N-terminal or C-terminal sequences may be provided forvarious reasons, it is often advantageous to include an additional aminoacid sequence which contains secretory or leader sequences, a pre-, pro-or prepro-protein sequence, or a sequence which aids in purification,such as an affinity tag. An additional sequence which may providestability during recombinant production may also be used. Such sequencesmay be optionally removed as required by incorporating a cleavablesequence as an additional sequence or part thereof. Thus an NKCC1polypeptide may be fused to other moieties including other polypeptides.Such additional sequences and affinity tags are well known in the art.Techniques for providing such additional sequences are well known in theart.

Additional sequences may be provided in order to alter thecharacteristics of a particular polypeptide. This can be useful inimproving expression or regulation of expression in particularexpression systems. For example, an additional sequence may provide someprotection against proteolytic cleavage.

Additional sequences can also be useful in altering the properties of apolypeptide to aid in identification or purification. For example, afusion protein may be provided in which a polypeptide is linked to amoiety capable of being isolated by affinity chromatography, forexample, but without limitation, multiple histidine residues, a FLAGtag, HA tag or myc tag. The moiety may be an antigen or an epitope andthe affinity column may comprise immobilised antibodies or immobilisedantibody fragments which bind to said antigen or epitope (desirably witha high degree of specificity). The fusion protein can usually be elutedfrom the column by addition of an appropriate buffer.

Additional N-terminal or C-terminal sequences may, however, be presentsimply as a result of a particular technique used to obtain apolypeptide and need not provide any particular advantageouscharacteristic to the polypeptide. Such polypeptides are within thescope of the present invention.

Whatever additional N-terminal or C-terminal sequence is present, it ispreferred that the resultant polypeptide should exhibit theimmunological or biological activity of the polypeptide having the aminoacid sequence shown in FIG. 1 (SEQ ID NO: 1).

Polypeptides within the Scope of b)

Turning now to the polypeptides defined in b) above, it will beappreciated by the person skilled in the art that these polypeptides arederivatives of the polypeptide given in a) above, provided that suchderivatives preferably exhibit the immunological or biological activityof the polypeptide having the amino acid sequence shown in FIG. 1 (SEQID NO: 1). Alternatively, the biological activity of the polypeptide maybe altered. As such, it will be appreciated by one skilled in the artthat derivatives can include post-translational modifications, forexample but without limitation, phosphorylation, glycosylation andfarnesylation. Modifications include naturally occurring modifications,such as, without limitation, post-translational modifications, andnon-naturally occurring modifications such as may be introduced bymutagenesis.

Alterations in the amino acid sequence of a protein can occur which donot affect the activity of a protein. These include amino aciddeletions, insertions and substitutions and can result from alternativesplicing and/or the presence of multiple translation start sites andstop sites. Polymorphisms may arise as a result of the infidelity of thetranslation process. Thus changes in amino acid sequence may betolerated which do not affect the protein's biological or immunologicalactivity.

In further aspects of the present invention, for example therapy and/orprophylaxis of breast, lung and/or pancreatic cancer the use of dominantnegative or constitutively active NKCC1 polypeptides is contemplated.Therefore, in one embodiment, the deleted, inserted, modified orsubstituted amino acid(s) renders dominant negative activity upon thepeptide. In another embodiment, the deleted, inserted, modified orsubstituted amino acid(s) renders the polypeptide constitutively active.

The skilled person will appreciate that various changes can often bemade to the amino acid sequence of a polypeptide which has a particularactivity to produce derivatives (sometimes known as variants or“muteins”) having at least a proportion of said activity, and preferablyhaving a substantial proportion of said activity. Such derivatives ofthe polypeptides described in a) above are within the scope of thepresent invention and are discussed in greater detail below. Theyinclude allelic and non-allelic derivatives.

An example of a derivative of an NKCC1 polypeptide is a polypeptide asdefined in a) above, apart from the substitution of one or more aminoacids with one or more other amino acids. The skilled person is awarethat various amino acids have similar properties. One or more such aminoacids of a polypeptide can often be substituted by one or more othersuch amino acids without eliminating a desired activity of thatpolypeptide.

Thus, the amino acids glycine, alanine, valine, leucine and isoleucinecan often be substituted for one another (amino acids having aliphaticside chains). Of these possible substitutions, it is preferred thatglycine and alanine are used to substitute for one another (since theyhave relatively short side chains) and that valine, leucine andisoleucine are used to substitute for one another (since they havelarger aliphatic side chains which are hydrophobic).

Other amino acids which can often be substituted for one anotherinclude:

-   -   phenylalanine, tyrosine and tryptophan (amino acids having        aromatic side chains);    -   lysine, arginine and histidine (amino acids having basic side        chains);    -   aspartate and glutamate (amino acids having acidic side chains);    -   asparagine and glutamine (amino acids having amide side chains);    -   cysteine and methionine (amino acids having sulphur-containing        side chains); and    -   aspartic acid and glutamic acid can substitute for        phospho-serine and phospho-threonine, respectively (amino acids        with acidic side chains).

Substitutions of this nature are often referred to as “conservative” or“semi-conservative” amino acid substitutions.

Amino acid deletions or insertions may also be made relative to theamino acid sequence given in a) above. Thus, for example, amino acidswhich do not have a substantial effect on the biological and/orimmunological activity of the polypeptide, or at least which do noteliminate such activity, may be deleted. Such deletions can beadvantageous since the overall length and the molecular weight of apolypeptide can be reduced whilst still retaining activity. This canenable the amount of polypeptide required for a particular purpose to bereduced for example, dosage levels can be reduced.

Amino acid insertions relative to the sequence given in a) above canalso be made. This may be done to alter the properties of an NKCC1polypeptide (e.g. to assist in identification, purification orexpression, as explained above in relation to fusion proteins).

Amino acid changes relative to the sequence given in a) above can bemade using any suitable technique, e.g. by using site-directedmutagenesis (Hutchinson et al., 1978, J. Biol. Chem. 253:6551).

It should be appreciated that amino acid substitutions or insertions tothe polypeptide for use in the present invention can be made usingnaturally occurring or non-naturally occurring amino acids. Whether ornot natural or synthetic amino acids are used, it is preferred that onlyL-amino acids are present.

Whatever amino acid changes are made (whether by means of substitution,modification, insertion or deletion), preferred NKCC1 polypeptides haveat least 50% sequence identity with a polypeptide as defined in a)above, more preferably the degree of sequence identity is at least 75%,at least 80%, at least 85%. Sequence identities of at least 90%, atleast 95% or at least 98% are most preferred.

The term identity can be used to describe the similarity between twopolypeptide sequences. The degree of amino acid sequence identity can becalculated using a program such as “bestfit” (Smith and Waterman,Advances in Applied Mathematics, 482-489 (1981)) to find the bestsegment of similarity between any two sequences. The alignment is basedon maximising the score achieved using a matrix of amino acidsimilarities, such as that described by Schwarz and Dayhof (1979) Atlasof Protein Sequence and Structure, Dayhof, M. O., Ed pp 353-358.

A software package well known in the art for carrying out this procedureis the CLUSTAL program. It compares the amino acid sequences of twopolypeptides and finds the optimal alignment by inserting spaces ineither sequence as appropriate. The amino acid identity or similarity(identity plus conservation of amino acid type) for an optimal alignmentcan also be calculated using a software package such as BLASTX. Thisprogram aligns the largest stretch of similar sequence and assigns avalue to the fit. For any one pattern comparison, several regions ofsimilarity may be found, each having a different score. One skilled inthe art will appreciate that two polypeptides of different lengths maybe compared over the entire length of the longer fragment. Alternativelysmall regions may be compared. Normally sequences of the same length arecompared for a useful comparison to be made.

Where high degrees of sequence identity are present there will berelatively few differences in amino acid sequence. Thus for example theymay be less than 20, less than 10, or even less than 5 differences.

Polypeptides within the Scope of c)

As discussed supra, it is often advantageous to reduce the length of apolypeptide, preferably the resultant reduced length polypeptide stillhas a desired activity or can give rise to useful antibodies. Feature c)therefore covers fragments of polypeptides a) or b) above for use in thepresent invention.

As used herein, the term “fragment” refers to a polypeptide comprisingan amino acid sequence of at least 10 amino acid residues (preferably,at least 15 amino acid residues, at least 20 amino acid residues, atleast 25 amino acid residues, at least 40 amino acid residues, at least50 amino acid residues, at least 60 amino residues, at least 70 aminoacid residues, at least 80 amino acid residues, at least 90 amino acidresidues, at least 100 amino acid residues, at least 125 amino acidresidues, at least 150 amino acid residues, at least 175 amino acidresidues, at least 200 amino acid residues, at least 250 amino acidresidues, at least 300 amino acid residues, at least 350 amino acidresidues, at least 400 amino acid residues, at least 450 amino acidresidues, at least 500 amino acid residues, at least 550 amino acidresidues, at least 600 amino acid residues, at least 650 amino acidresidues, at least 700 amino acid residues, at least 750 amino acidresidues, at least 800 amino acid residues, at least 850 amino acidresidues, at least 900 amino acid residues, at least 950 amino acidresidues, at least 1000 amino acid residues, at least 1050 amino acidresidues, at least 1100 amino acid residues, at least 1150 amino acidresidues or at least 1200 amino acid residues) of the amino acidsequence of a parent polypeptide. Any given fragment of the NKCC1polypeptide may or may not possess the functional activity of the parentpolypeptide. A fragment has at least 70% identity over its length to theamino acid sequence shown in FIG. 1 (SEQ ID NO: 1), preferably it has atleast 75%, at least 80%, at least 85%, at least 90%, at least 95% or atleast 98% identity. A skilled person can determine whether or not aparticular fragment has activity.

An NKCC1 polypeptide may be useful as antigenic material, and may beused in the production of vaccines for treatment or prophylaxis ofbreast, lung and/or pancreatic cancer. Such material can be “antigenic”and/or “immunogenic”. Generally, “antigenic” is taken to mean that theprotein is capable of being used to raise antibodies or indeed iscapable of inducing an antibody response in a subject. “Immunogenic” istaken to mean that the protein is capable of eliciting an immuneresponse in a subject. Thus, in the latter case, the protein may becapable of not only generating an antibody response but, in addition,non-antibody based immune responses.

It is well known that it is possible to screen an antigenic protein orpolypeptide to identify epitopic regions, i.e. those regions which areresponsible for the protein or polypeptide's antigenicity orimmunogenicity. Methods well known to the skilled person can be used totest fragments and/or homologues and/or derivatives for antigenicity.Thus, the fragments of NKCC1 polypeptides may include one or more suchepitopic regions or be sufficiently similar to such regions to retaintheir antigenic/immunogenic properties. Thus, for fragments of NKCC1polypeptides the degree of identity is perhaps irrelevant, since theymay be 100% identical to a particular part of a protein or polypeptide,homologue or derivative as described herein. The key issue may be thatthe fragment retains the antigenic and/or immunogenic properties of theprotein from which it is derived.

Homologues, derivatives and fragments may possess at least a degree ofthe antigenicity and/or immunogenicity of the protein from which theyare derived.

As will be discussed below, NKCC1 polypeptides find use in a therapeuticapproach to breast, lung and/or pancreatic cancer. In a particularembodiment, an NKCC1 polypeptide is fused to another polypeptide, suchas the protein transduction domain of the HIV/Tat protein, whichfacilitates the entry of fusion protein into a cell (Asob, S. et al.,2002, Proc. Natl. Acad. Sci. USA, 99:17107-17112). Such a fusion proteinmay be provided for use in the preparation of a composition for thetreatment of breast, lung and/or pancreatic cancer. The skilled personwill appreciate that for the preparation of one or more suchpolypeptides, the preferred approach will be based on recombinant DNAtechniques.

The NKCC1 polypeptides can be coded for by a large variety of nucleicacid molecules, taking into account the well-known degeneracy of thegenetic code. All of these molecules can be used in the presentinvention. They can be inserted into vectors and cloned to provide largeamounts of DNA or RNA for further study. Suitable vectors may beintroduced into host cells to enable the expression of polypeptides usedin the present invention using techniques known to the person skilled inthe art.

Techniques for cloning, expressing and purifying proteins andpolypeptides are well known to the skilled person. DNA constructs canreadily be generated using methods well known in the art. Thesetechniques are disclosed, for example in J. Sambrook et al, MolecularCloning 2^(nd) Edition, Cold Spring Harbour Laboratory Press (1989); inOld & Primrose Principles of Gene Manipulation 5th Edition, BlackwellScientific Publications (1994); and in Stryer Biochemistry 4th Edition,W H Freeman and Company (1995). Modifications of DNA constructs and theproteins expressed such as the addition of promoters, enhancers, signalsequences, leader sequences, translation start and stop signals and DNAstability controlling regions, or the addition of fusion partners maythen be facilitated.

Normally the DNA construct will be inserted into a vector, which may beof phage or plasmid origin. Expression of the protein is achieved by thetransformation or transfection of the vector into a host cell, which maybe of eukaryotic or prokaryotic origin. Such vectors and suitable hostcells form further aspects for use in the present invention.

For recombinant NKCC1 polypeptide production, host cells can begenetically engineered to incorporate expression systems or portionsthereof for NKCC1 nucleic acids. Such incorporation can be performedusing methods well known in the art, such as, calcium phosphatetransfection, DEAE-dextran mediated transfection, transvection,microinjection, cationic lipid mediated transfection, electroporation,transduction, scrape loading, ballistic introduction or infection (seee.g. Davis et al., Basic Methods in Molecular Biology, 1986 and Sambrooket al., Molecular Cloning: a Laboratory Manual, 2^(nd) Ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Representative examples of host cells include bacterial cells e.g. E.Coli, Streptococci, Staphylococci, Streptomyces and Bacillus subtiliscells; fungal cells, such as yeast cells and Aspergillus cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS, HeLa, C127, 3T3, HEK 293, BHK and Bowes melanoma cells; andplant cells.

A wide variety of expression systems can be used, such as and withoutlimitation, chromosomal, episomal and virus-derived systems, e.g.vectors derived from bacterial plasmids, from bacteriophage, fromtransposons, from yeast episomes, from insertion elements, from yeastchromosomal elements, from viruses such as baculoviruses, papova virusessuch as SV40, vaccinia viruses, adenoviruses, fowl pox viruses,pseudorabies viruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Theexpression systems may contain control regions that regulate as well asengender expression. Generally, any system or vector which is able tomaintain, propagate or express a nucleic acid to produce a polypeptidein a host may be used. The appropriate nucleic acid sequence may beinserted into an expression system by any variety of well-known androutine techniques, such as those set forth in Sambrook et al., supra.Appropriate secretion signals may be incorporated into the NKCC1polypeptide to allow secretion of the translated protein into the lumenof the endoplasmic reticulum, the periplasmic space or the extracellularenvironment. These signals may be endogenous to the NKCC1 polypeptide orthey may be heterologous signals.

Cell-free translation systems can also be employed to producerecombinant polypeptides (e.g. rabbit reticulocyte lysate, wheat germlysate, SP6/T7 in vitro T & T and RTS 100 E. Coli HY coupledTranscription/Translation kits from Roche Diagnostics Ltd., Lewes, UKand the TNT Quick coupled Transcription/Translation System from PromegaUK, Southampton, UK.

The NKCC1 nucleic acids may be synthesised using methods known in theart, such as using conventional chemical approaches or polymerase chainreaction (PCR) amplification. The NKCC1 nucleic acids also permit theidentification and cloning of the gene encoding an NKCC1 polypeptidefrom any species, for instance by screening cDNA libraries, genomiclibraries or expression libraries.

Knowledge of the nucleic acid structure can be used to raise antibodiesand for gene therapy. Techniques for this are well-known by thoseskilled in the art, as discussed in more detail herein.

By using appropriate expression systems, NKCC1 polypeptides may beexpressed in glycosylated or non-glycosylated form. Non-glycosylatedforms can be produced by expression in prokaryotic hosts, such as E.coli.

Polypeptides comprising N-terminal methionine may be produced usingcertain expression systems, whilst in others the mature polypeptide willlack this residue.

Preferred techniques for cloning, expressing and purifying an NKCC1polypeptide are summarised below:

Polypeptides may be prepared natively or under denaturing conditions andthen subsequently refolded. Baculoviral expression vectors includesecretory plasmids (such as pACGP67 from Pharmingen), which may have anepitope tag sequence cloned in frame (e.g. myc, V5 or His) to aiddetection and allow for subsequent purification of the protein.Mammalian expression vectors may include pCDNA3 and pSecTag (bothInvitrogen), and pREP9 and pCEP4 (Invitrogen). E. coli systems includethe pBad series (His tagged-Invitrogen) or pGex series (Pharmacia).

In one embodiment, NKCC1 polypeptides are provided in isolated form andinclude NKCC1 polypeptides that have been purified to at least someextent. NKCC1 polypeptides can be produced using recombinant methods,synthetically produced or produced by a combination of these methods.NKCC1 polypeptides may be provided in substantially pure form, that isto say free, to a substantial extent, from other proteins.

If an NKCC1 polypeptide is to be expressed for use in cell-basedscreening assays, as described below, it is preferred that thepolypeptide is produced at the cell surface. In this event, the cellsmay be harvested prior to use in the screening assay. If the NKCC1polypeptide is secreted into the medium, the medium can be recovered inorder to isolate said polypeptide. If produced intracellularly, thecells must first be lysed before the NKCC1 polypeptide is recovered.

NKCC1 polypeptides can be recovered and purified from recombinant cellcultures by well-known methods including, ammonium sulphate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, affinity chromatography, hydrophobicinteraction chromatography, hydroxylapatite chromatography, molecularsieving chromatography, centrifugation methods, electrophoresis methodsand lectin chromatography. In one embodiment, a combination of thesemethods is used. In another embodiment, high performance liquidchromatography is used. In a further embodiment, an antibody whichspecifically binds to an NKCC1 polypeptide can be used to deplete asample, comprising an NKCC1 polypeptide, of said polypeptide or topurify said polypeptide. Techniques well-known in the art, may be usedfor refolding to regenerate native or active conformations of the NKCC1polypeptides when the polypeptides have been denatured during isolationand or purification.

In addition to nucleic acid molecules coding for NKCC1 polypeptides,referred to herein as “coding” nucleic acids, the present invention alsoutilises nucleic acids complementary thereto. Thus, for example, bothstrands of a double stranded nucleic acid molecule are included in thepresent invention (whether or not they are associated with one another).Also included are mRNA molecules and complementary DNA molecules (e.g.cDNA molecules).

The use of nucleic acid molecules which can hybridise to any of thenucleic acid molecules discussed above, in the diagnosis, screening,treatment and/or prophylaxis of breast, lung and/or pancreatic cancer isalso covered by the present invention. Such nucleic acid molecules arereferred to herein as “hybridising” nucleic acid molecules. Hybridisingnucleic acid molecules can be useful as probes or primers, for example.

Desirably such hybridising molecules are at least 8 nucleotides inlength and preferably are at least 25 or at least 50 nucleotides inlength. The hybridising nucleic acid molecules preferably hybridise tonucleic acids within the scope of d), e), f), g), h) or i) abovespecifically.

Desirably the hybridising molecules will hybridise to such moleculesunder stringent hybridisation conditions. One example of stringenthybridisation conditions is where attempted hybridisation is carried outat a temperature of from about 35° C. to about 65° C. using a saltsolution that is about 0.9 molar. However, the skilled person will beable to vary such conditions as appropriate in order to take intoaccount variables such as probe length, base composition, type of ionspresent, etc. For a high degree of selectivity, relatively stringentconditions are used to form the duplexes, such as low salt or hightemperature conditions. As used herein, “highly stringent conditions”means hybridisation to filter-bound DNA in 0.5 M NaHPO₄, 7% sodiumdodecyl sulphate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1%SDS at 68° C. (Ausubel P. M. et al., eds., 1989, Current Protocols inMolecular Biology, Vol. I, Green Publishing Associates, Inc., and JohnWiley & Sons, Inc., New York, at p. 2.10.3). For some applications, lessstringent conditions for duplex formation are required. As used herein“moderately stringent conditions” means washing in 0.2×SSC/0.1% SDS at42° C. (Ausubel et al., 1989, supra). Hybridisation conditions can alsobe rendered more stringent by the addition of increasing amounts offormamide, to destabilise the hybrid duplex. Thus, particularhybridisation conditions can be readily manipulated, and will generallybe chosen depending on the desired results. In general, convenienthybridisation temperatures in the presence of 50% formamide are: 42° C.for a probe which is 95 to 100% identical to the fragment of a geneencoding an NKCC1 polypeptide, 37° C. for 90 to 95% identity and 32° C.for 70 to 90% identity. In the preparation of genomic libraries, DNAfragments are generated, some of which will encode parts or the whole ofan NKCC1 polypeptide. The DNA may be cleaved at specific sites usingvarious restriction enzymes. Alternatively, one may use DNAse in thepresence of manganese to fragment the DNA, or the DNA can be physicallysheared, for example, by sonication. The DNA fragments can then beseparated according to size by standard techniques, including but notlimited to agarose and polyacrylamide gel electrophoresis, columnchromatography and sucrose gradient centrifugation. The DNA fragmentscan then be inserted into suitable vectors, including but not limited toplasmids, cosmids, bacteriophages lambda or T₄, and yeast artificialchromosomes (YACs). (See, for example, Sambrook et al., 1989, MolecularCloning, A Laboratory Manual, 1D Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.; Glover, D. M. (ed.), 1985, DNA Cloning:A Practical Approach, MRL Press, Ltd, Oxford, U.K. Vol. I, II; AusubelF. M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol.I, Green Publishing Associates, Inc., and John Wiley & sons, Inc., NewYork). The genomic library may be screened by nucleic acid hybridisationto labelled probe (Benton & Davis, 1977, Science 196:180; Grunstein &Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961).

Manipulation of the DNA encoding a protein is a particularly powerfultechnique for both modifying proteins and for generating largequantities of protein for purification purposes. This may involve theuse of PCR techniques to amplify a desired nucleic acid sequence. Thusthe sequence data provided herein can be used to design primers for usein PCR so that a desired sequence can be targeted and then amplified toa high degree.

Typically, primers will be at least eight nucleotides long and willgenerally be at least ten nucleotides long (e.g. fifteen to twenty-fivenucleotides long). In some cases, primers of at least thirty or at leastthirty-five nucleotides in length may be used.

As a further alternative chemical synthesis may be used, this may beautomated. Relatively short sequences may be chemically synthesised andligated together to provide a longer sequence.

The term identity can also be used to describe the similarity betweentwo individual DNA sequences. The ‘bestfit’ program (Smith and Waterman,1981, Advances in applied Mathematics, 482-489) is one example of a typeof computer software used to find the best segment of similarity betweentwo nucleic acid sequences, whilst the GAP program enables sequences tobe aligned along their whole length and finds the optimal alignment byinserting spaces in either sequence as appropriate. It is preferred ifsequences which show substantial identity with any of those of d), e)and f) have e.g. at least 50%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95% or at least 98% sequence identity.

A hybridising nucleic acid molecule of use in the present invention mayhave a high degree of sequence identity along its length with a nucleicacid molecule within the scope of d)-i) above (e.g. at least 50%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95% or atleast 98% sequence identity). As will be appreciated by the skilledperson, the higher the sequence identity a given single stranded nucleicacid molecule has with another nucleic acid molecule, the greater thelikelihood that it will hybridise to a nucleic acid molecule which iscomplementary to that other nucleic acid molecule under appropriateconditions.

If desired, a gene encoding an NKCC1 polypeptide, a related gene, orrelated nucleic acid sequences or subsequences, including complementarysequences, can also be, used in hybridisation assays. A nucleotideencoding an NKCC1 polypeptide, or subsequences thereof comprising atleast 8 nucleotides, can be used as a hybridisation probe. Hybridisationassays can be used for detection, prognosis, diagnosis, or monitoring ofconditions, disorders, or disease states, associated with aberrantexpression of genes encoding an NKCC1 polypeptide, or for differentialdiagnosis of patients with signs or symptoms suggestive of breast, lungand/or pancreatic cancer.

In particular embodiment, such a hybridisation assay can be carried outby a method comprising:

-   -   i) contacting a biological sample containing nucleic acid with a        nucleic acid probe capable of hybridising to a DNA or RNA that        encodes an NKCC1 polypeptide, under conditions such that        hybridisation can occur; and    -   ii) detecting or measuring any resulting hybridisation.

The invention also provides a kit comprising a nucleic acid probecapable of hybridising to RNA encoding an NKCC1 polypeptide. In aspecific embodiment, a kit comprises in one or more containers a pair ofprimers (e.g. each in the size range of 6-30 nucleotides, morepreferably 10-30 nucleotides and still more preferably 10-20nucleotides) that under appropriate reaction conditions can primeamplification of at least a portion of a nucleic acid encoding an NKCC1polypeptide, such as by polymerase chain reaction (see e.g. Innis etal., 1990, PCR Protocols, Academic Press, Inc., San Diego, Calif.),ligase chain reaction (see EP 320308) use of Qβ replicase, cyclic probereaction, or other methods known in the art.

In another embodiment, a preparation of oligonucleotides comprising 10or more consecutive nucleotides complementary to a nucleotide sequenceencoding an NKCC1 polypeptide for use as vaccines for the treatment ofbreast, lung and/or pancreatic cancer. Such preparations may includeadjuvants or other vehicles.

In yet another embodiment, the present invention provides the use of atleast one NKCC1 nucleic acid in the preparation of a pharmaceuticalcomposition for use in the treatment of breast, lung and/or pancreaticcancer.

In addition to being used as primers and/or probes, hybridising nucleicacid molecules can be used as anti-sense molecules to alter theexpression of NKCC1 polypeptides by binding to complementary nucleicacid molecules. This technique can be used in antisense therapy.

As used herein, an “anti-sense” nucleic acid refers to a nucleic acidcapable of hybridising by virtue of some sequence complementarity to aportion of an RNA (preferably mRNA) encoding a polypeptide as definedherein. The antisense nucleic acid may be complementary to a codingand/or non-coding region of a mRNA encoding such a polypeptide. Suchantisense nucleic acids have utility as compounds that inhibitexpression, and can be used in the treatment or prevention of breast,lung and/or pancreatic cancer.

In a specific embodiment, expression of an NKCC1 polypeptide isinhibited by use of antisense nucleic acids. The present inventionprovides the therapeutic or prophylactic use of nucleic acids comprisingat least eight nucleotides that are antisense to a gene or cDNA encodingan NKCC1 polypeptide.

Endogenous polypeptide expression can also be reduced by inactivating or“knocking out” the gene encoding the polypeptide, or the promoter ofsuch a gene, using targeted homologous recombination (e.g. see Smithies,et al., 1985, Nature 317:230-234; Thomas & Capecchi, 1987, Cell51:503-512; Thompson et al., 1989, Cell 5:313-321; and Zijlstra et al.,1989, Nature 342:435-438). For example, a mutant gene encoding anon-functional polypeptide (or a completely unrelated DNA sequence)flanked by DNA homologous to the endogenous gene (either the codingregions or regulatory regions of the gene encoding the polypeptide) canbe used, with or without a selectable marker and/or a negativeselectable marker, to transfect cells that express the target gene invivo. Insertion of the DNA construct, via targeted homologousrecombination, results in inactivation of the target gene. This approachcan be adapted for use in humans, provided the recombinant DNAconstructs are directly administered or targeted to the required site invivo using appropriate viral vectors.

In another embodiment, symptoms of breast, pancreatic and/or lung cancermay be ameliorated by decreasing the level or activity of an NKCC1polypeptide by using gene sequences encoding an NKCC1 polypeptide inconjunction with well-known gene “knock-out”, ribozyme or triple helixmethods to decrease gene expression of the polypeptide. In thisapproach, ribozyme or triple helix molecules are used to modulate theactivity, expression or synthesis of the gene, and thus to amelioratethe symptoms of breast, pancreatic and/or lung cancer. Such moleculesmay be designed to reduce or inhibit expression of a mutant ornon-mutant target gene. Techniques for the production and use of suchmolecules are well known to those of skill in the art.

In a further embodiment, the NKCC1 nucleic acid is administered via genetherapy (see for example Hoshida, T. et al., 2002, Pancreas, 25:111-121;Ikuno, Y. 2002, Invest. Ophthalmol. Vis. Sci. 2002 43:2406-2411;Bollard, C., 2002, Blood 99:3179-3187; Lee E., 2001, Mol. Med.7:773-782).

Gene therapy refers to administration to a subject of an expressed orexpressible nucleic acid. In this embodiment, the nucleic acid producesits encoded protein that mediates a therapeutic effect by promotingpolypeptide function. Any of the methods for gene therapy available inthe art can be used according to the present invention.

In a preferred aspect, the pharmaceutical composition comprises an NKCC1nucleic acid, such as a nucleic acid encoding an NKCC1 polypeptide, saidnucleic acid being part of an expression vector that expresses an NKCC1polypeptide or chimeric protein thereof in a suitable host. Inparticular, such a nucleic acid has a promoter operably linked to thepolypeptide coding region, said promoter being inducible or constitutive(and, optionally, tissue-specific). In another particular embodiment, anucleic acid molecule is used in which the coding sequences and anyother desired sequences are flanked by regions that promote homologousrecombination at a desired site in the genome, thus providing forintrachromosomal expression of the nucleic acid (Koller & Smithies,1989, Proc. Natl. Acad. Sci USA 86:8932-8935; Zijlstra et al., 1989,Nature 342:435-438).

Delivery of the nucleic acid into a patient may be direct, in which casethe patient is directly exposed to the nucleic acid or nucleicacid-carrying vector; this approach is known as in vivo gene therapy.Alternatively, delivery of the nucleic acid into the patient may beindirect, in which case cells are first transformed with the nucleicacid in vitro and then transplanted into the patient; this approach isknown as ex vivo gene therapy.

The present invention also demonstrates that NKCC1 is a suitableimmunotherapeutic target for the treatment and/or prophylaxis of breast,pancreatic and/or lung cancer. Therefore, in a further aspect thepresent invention provides antibodies that recognise NKCC1 polypeptidesand their use in the treatment and/or prophylaxis of breast, pancreaticand/or lung cancer.

NKCC1 polypeptides, may be used as immunogens to generate antibodieswhich immunospecifically bind such an immunogen, these are referred toherein as NKCC1 antibodies. NKCC1 antibodies include, but are notlimited to polyclonal, monoclonal, bispecific, humanised or chimericantibodies, single chain antibodies, Fab fragments and F(ab′) fragments,fragments produced by a Fab expression library, anti-idiotypic (anti-Id)antibodies, and epitope-binding fragments of any of the above. The term“antibody” as used herein refers to immunoglobulin molecules andimmunologically-active portions of immunoglobulin molecules, i.e.molecules that contain an antigen binding site that specifically bindsan antigen. The immunoglobulin molecules can be of any class (e.g. IgG,IgE, IgM, IgD and IgA) or subclass of immunoglobulin molecule. Preferredantibodies bind specifically to NKCC1 polypeptides so that they can beused to purify and/or inhibit the activity of such polypeptides.Specifically recognising or binding specifically means that theantibodies have a greater affinity for NKCC1 polypeptides than for otherpolypeptides.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g. ELISA(enzyme-linked immunosorbent assay). For example, to select antibodies,which recognise a specific domain of an NKCC1 polypeptide, one may assaygenerated hybridomas for a product which binds to a polypeptide fragmentcontaining such domain. For selection of an antibody that specificallybinds a first polypeptide homologue but which does not specifically bindto (or binds less avidly to) a second polypeptide homologue, one canselect on the basis of positive binding to the first polypeptidehomologue and a lack of binding to (or reduced binding to) the secondpolypeptide homologue.

For preparation of NKCC1 monoclonal antibodies (mAbs), any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture may be used. For example, the hybridoma techniqueoriginally developed by Kohler and Milstein (1975, Nature 256:495-497),as well as the trioma technique, the human B-cell hybridoma technique(Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridomatechnique to produce human monoclonal antibodies (Cole et al., 1985, inMonoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAbs for use in the invention may be cultivated in vitroor in vivo. In an additional embodiment of the invention, mAbs can beproduced in germ-free animals utilising known technology.

The mAbs include but are not limited to human mAbs and chimeric mAbs(e.g. human-mouse chimeras). A chimeric antibody is a molecule in whichdifferent portions are derived from different animal species, such asthose having a human immunoglobulin constant region and a variableregion derived from a murine mAb, (see, e.g. U.S. Pat. No. 4,816,567;and U.S. Pat. No. 4,816,397). Humanised antibodies are antibodymolecules from non-human species having one or more complementaritydetermining regions (CDRs) from the non-human species and a frameworkregion from a human immunoglobulin molecule, (see, e.g. U.S. Pat. No.5,585,089).

Chimeric and humanised mAbs can be produced by recombinant DNAtechniques known in the art, for example using methods described in WO87/02671; EP 184187; EP 171496; EP 173494; WO 86/01533; U.S. Pat. No.4,816,567; EP 125,023; Better et al., 1988, Science 240:1041-1043; Liuet al., 1987, Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987,J. Immunol. 139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al.,1985, Nature 314:446-449; Shaw et al., 1988, J. Natl. Cancer Inst.80:1553-1559; Morrison, 1985, Science 229:1202-1207; Oi et al., 1986,Bio/Techniques 4:214; U.S. Pat. No. 5,225,539; Jones et al., 1986,Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al., 1988, J. Immunol. 141:4053-4060.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Such antibodies can be produced usingtransgenic mice which are incapable of expressing endogenousimmunoglobulin heavy and light chain genes, but which can express humanheavy and light chain genes. The transgenic mice are immunised in thenormal fashion with a selected antigen, e.g. all or a portion of apolypeptide for use in the invention. mAbs directed against the antigencan be obtained using conventional hybridoma technology. The humanimmunoglobulin transgenes harboured by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM, IgD and IgE antibodies.For an overview of this technology for producing human antibodies, seeLonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g. U.S. Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No.5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806.

Completely human antibodies, which recognise a selected epitope, can begenerated using a technique referred to as “guided selection”. In thisapproach a selected non-human monoclonal antibody, e.g. a mouseantibody, is used to guide the selection of a completely human antibodyrecognising the same epitope, (Jespers et al. (1994) Bio/technology12:899-903).

The NKCC1 antibodies can also be generated using various phage displaymethods known in the art. In phage display methods, functional antibodydomains are displayed on the surface of phage particles which carry thepolynucleotide sequences encoding them. In a particular, such phage canbe utilised to display antigen binding domains expressed from arepertoire or combinatorial antibody library (e.g. human or murine).Phage expressing an antigen binding domain that binds the antigen ofinterest can be selected or identified with antigen, e.g. using labelledantigen or antigen bound or captured to a solid surface or bead. Phageused in these methods are typically filamentous phage including fd andM13 binding domains expressed from phage with Fab, Fv or disulphidestabilised Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Phage display methods that can be used tomake the antibodies of the present invention include those disclosed inBrinkman et al., J. Immunol. Methods 182: 41-50 (1995); Ames et al., J.Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J.Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burtonet al., Advances in Immunology 57:191-280 (1994); WO 90/02809; WO91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717;5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637;5,780,225; 5,658,727; 5,733,743 and 5,969,108.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g. as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in WO92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawaiet al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043(1988).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. No. 4,946,778, U.S. Pat.No. 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991);Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science240:1038-1040 (1988).

The invention further provides for the use of bispecific antibodies,which can be made by methods known in the art. Traditional production offull length bispecific antibodies is based on the coexpression of twoimmunoglobulin heavy chain-light chain pairs, where the two chains havedifferent specificities (Milstein et al., 1983, Nature 305:537-539).Because of the random assortment of immunoglobulin heavy and lightchains, these hybridomas (quadromas) produce a potential mixture of 10different antibody molecules, of which only one has the correctbispecific structure. Purification of the correct molecule, which isusually done by affinity chromatography steps, is rather cumbersome, andthe product yields are low. Similar procedures are disclosed in WO93/08829, and in Traunecker et al., 1991, EMBO J. 10:3655-3659.

According to a different and more preferred approach, antibody variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant domain sequences.The fusion preferably is with an immunoglobulin heavy chain constantdomain, comprising at least part of the hinge, CH2, and CH3 regions. Itis preferred to have the first heavy-chain constant region (CH1)containing the site necessary for light chain binding, present in atleast one of the fusions. DNAs encoding the immunoglobulin heavy chainfusions and, if desired, the immunoglobulin light chain, are insertedinto separate expression vectors, and are co-transfected into a suitablehost organism. This provides for great flexibility in adjusting themutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm.This asymmetric structure facilitates the separation of the desiredbispecific compound from unwanted immunoglobulin chain combinations, asthe presence of an immunoglobulin light chain in only one half of thebispecific molecule provides for a facile way of separation. Thisapproach is disclosed in WO 94/04690. For further details for generatingbispecific antibodies see, for example, Suresh et al., Methods inEnzymology, 1986, 121:210.

The invention provides functionally-active fragments, derivatives oranalogues of the anti-NKCC1 antibodies. “Functionally-active” means thatthe fragment, derivative or analogue is able to elicitanti-anti-idiotype antibodies (i.e. tertiary antibodies) that recognisethe same antigen that is recognised by the antibody from which thefragment, derivative or analogue is derived. Specifically, in apreferred embodiment, the antigenicity of the idiotype of theimmunoglobulin molecule may be enhanced by deletion of framework and CDRsequences that are C-terminal to the CDR sequence that specificallyrecognises the antigen. To determine which CDR sequences bind theantigen, synthetic peptides containing the CDR sequences can be used inbinding assays with the antigen by any binding assay method known in theart.

The present invention provides antibody fragments such as, but notlimited to, F(ab′)2 fragments and Fab fragments. Antibody fragmentswhich recognise specific epitopes may be generated by known techniques.F(ab′)2 fragments consist of the variable region, the light chainconstant region and the CH1 domain of the heavy chain and are generatedby pepsin digestion of the antibody molecule. Fab fragments aregenerated by reducing the disulphide bridges of the F(ab′)2 fragments.The invention also provides heavy chain and light chain dimers of theNKCC1 antibodies, or any minimal fragment thereof such as Fvs or singlechain antibodies (SCAs) (e.g. as described in U.S. Pat. No. 4,946,778;Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad.Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54), or anyother molecule with the same specificity as the NKCC1 antibody. Singlechain antibodies are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge, resulting in asingle chain polypeptide. Techniques for the assembly of functional Fvfragments in E. coli may be used (Skerra et al., 1988, Science242:1038-1041).

In other embodiments, the invention provides fusion proteins of theNKCC1 antibodies (or functionally active fragments thereof), for examplein which the antibody is fused via a covalent bond (e.g. a peptidebond), at either the N-terminus or the C-terminus to an amino acidsequence of another protein (or portion thereof, preferably at least a10, 20 or 50 amino acid portion of the protein) that is not theantibody. Preferably the antibody, or fragment thereof, is covalentlylinked to the other protein at the N-terminus of the constant domain. Asstated above, such fusion proteins may facilitate purification, increasehalf-life in vivo, and enhance the delivery of an antigen across anepithelial barrier to the immune system.

The NKCC1 antibodies include analogues and derivatives that aremodified, e.g. by the covalent attachment of any type of molecule, aslong as such covalent attachment that does not impair immunospecificbinding. For example, but not by way of limitation, the derivatives andanalogues of the antibodies include those that have been furthermodified, e.g. by glycosylation, acetylation, pegylation, phosphylation,amidation, derivatisation by known protecting/blocking groups,proteolytic cleavage, linkage to a cellular ligand or other protein,etc. Any of numerous chemical modifications may be carried out by knowntechniques, including, but not limited to specific chemical cleavage,acetylation, formylation, etc. Additionally, the analogue or derivativemay contain one or more non-classical amino acids.

The foregoing antibodies can be used in methods known in the artrelating to the localisation and activity of the NKCC1 polypeptides,e.g. for imaging or radioimaging these proteins, measuring levelsthereof in appropriate physiological samples, in diagnostic methods,etc. and for radiotherapy.

The NKCC1 antibodies can be produced by any method known in the art forthe synthesis of antibodies, in particular, by chemical synthesis or byrecombinant expression, and are preferably produced by recombinantexpression technique.

Recombinant expression of antibodies, or fragments, derivatives oranalogues thereof, requires construction of a nucleic acid that encodesthe antibody. If the nucleotide sequence of the antibody is known, anucleic acid encoding the antibody may be assembled from chemicallysynthesised oligonucleotides (e.g. as described in Kutmeier et al.,1994, BioTechniques 17:242), which, briefly, involves the synthesis ofoverlapping oligonucleotides containing portions of the sequenceencoding antibody, annealing and ligation of those oligonucleotides, andthen amplification of the ligated oligonucleotides by PCR.

Alternatively, the nucleic acid encoding the antibody may be obtained bycloning the antibody. If a clone containing the nucleic acid encodingthe particular antibody is not available, but the sequence of theantibody molecule is known, a nucleic acid encoding the antibody may beobtained from a suitable source (e.g. an antibody cDNA library, or cDNAlibrary generated from any tissue or cells expressing the antibody) byPCR amplification using synthetic primers hybridisable to the 3′ and 5′ends of the sequence or by cloning using an oligonucleotide probespecific for the particular gene sequence.

If an antibody molecule that specifically recognises a particularantigen is not available (or a source for a cDNA library for cloning anucleic acid encoding such an antibody), antibodies specific for aparticular antigen may be generated by any method known in the art, forexample, by immunising an animal, such as a rabbit, to generatepolyclonal antibodies or, more preferably, by generating monoclonalantibodies. Alternatively, a clone encoding at least the Fab portion ofthe antibody may be obtained by screening Fab expression libraries (e.g.as described in Huse et al., 1989, Science 246:1275-1281) for clones ofFab fragments that bind the specific antigen or by screening antibodylibraries (See, e.g. Clackson et al., 1991, Nature 352:624; Hane et al.,1997 Proc. Natl. Acad. Sci. USA 94:4937).

Once a nucleic acid encoding at least the variable domain of theantibody molecule is obtained, it may be introduced into a vectorcontaining the nucleotide sequence encoding the constant region of theantibody molecule (e.g. WO 86/05807; WO 89/01036; and U.S. Pat. No.5,122,464). Vectors containing the complete light or heavy chain forco-expression with the nucleic acid to allow the expression of acomplete antibody molecule are also available. Then, the nucleic acidencoding the antibody can be used to introduce the nucleotidesubstitution(s) or deletion(s) necessary to substitute (or delete) theone or more variable region cysteine residues participating in anintrachain disulphide bond with an amino acid residue that does notcontain a sulphydryl group. Such modifications can be carried out by anymethod known in the art for the introduction of specific mutations ordeletions in a nucleotide sequence, for example, but not limited to,chemical mutagenesis, in vitro site directed mutagenesis (Hutchinson etal., 1978, J. Biol. Chem 253:6551), PCR based methods, etc.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855;Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature314:452-454) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human antibodyconstant region, e.g. humanised antibodies.

Once a nucleic acid encoding an antibody molecule has been obtained, thevector for the production of the antibody molecule may be produced byrecombinant DNA technology using techniques well known in the art. Thus,methods for preparing these antibodies by expressing a nucleic acidcontaining the antibody molecule sequences are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing an antibody molecule codingsequences and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination.See, for example, the techniques described in Sambrook et al. (1990,Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.) and Ausubel et al. (eds., 1998,Current Protocols in Molecular Biology, John Wiley & Sons, NY).

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an NKCC1 antibody.

The host cells used to express a recombinant NKCC1 antibody may beeither bacterial cells such as Escherichia coli, or, preferably,eukaryotic cells, especially for the expression of whole recombinantantibody molecule. In particular, mammalian cells such as Chinesehamster ovary cells (CHO), in conjunction with a vector such as themajor intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., 1986, Gene 45(1): pp 101-5; Cockett et al., 1990,BioTechnology 8:2).

A variety of host-expression vector systems may be utilised to expressan NKCC1 antibody molecule. Such host-expression systems representvehicles by which the coding sequences of interest may be produced andsubsequently purified, but also represent cells which may, whentransformed or transfected with the appropriate nucleotide codingsequences, express the NKCC1 antibody in situ. These include but are notlimited to microorganisms such as bacteria (e.g. E. coli, B. subtilis)transformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors containing antibody coding sequences; yeast (e.g.Saccharomyces, Pichia) transformed with recombinant yeast expressionvectors containing antibody coding sequences; insect cell systemsinfected with recombinant virus expression vectors (e.g. baculovirus)containing the antibody coding sequences; plant cell systems infectedwith recombinant virus expression vectors (e.g. cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinantplasmid expression vectors (e.g. Ti plasmid) containing antibody codingsequences; or mammalian cell systems (e.g. COS, CHO, BHK, HEK 293, 3T3cells) harbouring recombinant expression constructs containing promotersderived from the genome of mammalian cells (e.g. metallothioneinpromoter) or from mammalian viruses (e.g. the adenovirus late promoter;the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions comprising an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J.2:1791), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985,Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol.Chen. 24:5503-5509); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding to amatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example, the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample, the polyhedrin promoter). In mammalian host cells, a number ofviral-based expression systems (e.g. an adenovirus expression system)may be utilised.

As discussed above, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.glycosylation) and processing (e.g. cleavage) of protein products may beimportant for the activity of the protein.

For long-term, high-yield production of recombinant antibodies, stableexpression is preferred. For example, cells lines that stably express anantibody of interest can be produced by transfecting the cells with anexpression vector comprising the nucleotide sequence of the antibody andthe nucleotide sequence of a selectable marker (e.g. neomycin orhygromycin), and selecting for expression of the selectable marker. Suchengineered cell lines may be particularly useful in screening andevaluation of compounds that interact directly or indirectly with theantibody molecule.

The expression levels of the antibody molecule can be increased byvector amplification (for a review, see Bebbington and Hentschel, “Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning”, Vol. 3. (Academic Press, NewYork, 1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol.3:257).

The host cell may be co-transfected with two expression vectors, thefirst vector encoding a heavy chain derived polypeptide and the secondvector encoding a light chain derived polypeptide. The two vectors maycontain identical selectable markers which enable equal expression ofheavy and light chain polypeptides. Alternatively, a single vector maybe used which encodes both heavy and light chain polypeptides. In suchsituations, the light chain should be placed before the heavy chain toavoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197). The codingsequences for the heavy and light chains may comprise cDNA or genomicDNA.

Once the NKCC1 antibody has been recombinantly expressed, it may bepurified by any method known in the art for purification of an antibodymolecule, for example, by chromatography (e.g. ion exchangechromatography, affinity chromatography such as with protein A orspecific antigen, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins.

Alternatively, any fusion protein may be readily purified by utilisingan antibody specific for the fusion protein being expressed. Forexample, a system described by Janknecht et al. allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897).In this system, the gene of interest is subcloned into a vacciniarecombination plasmid such that the open reading frame of the gene istranslationally fused to an amino-terminal tag consisting of sixhistidine residues. The tag serves as a matrix binding domain for thefusion protein. Extracts from cells infected with recombinant vacciniavirus are loaded onto Ni²⁺ nitriloacetic acid-agarose columns andhistidine-tagged proteins are selectively eluted withimidazole-containing buffers.

In a preferred embodiment, NKCC1 antibodies are conjugated to adiagnostic or therapeutic moiety. The antibodies can be used fordiagnosis or to determine the efficacy of a given treatment regimen.Detection can be facilitated by coupling the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, radioactive nuclides, positron emitting metals(for use in positron emission tomography), and nonradioactiveparamagnetic metal ions. See generally U.S. Pat. No. 4,741,900 for metalions which can be conjugated to antibodies for use as diagnosticsaccording to the present invention. Suitable enzymes include horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; suitable prosthetic groups include streptavidin,avidin and biotin; suitable fluorescent materials include umbelliferone,fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin;suitable luminescent materials include luminol; suitable bioluminescentmaterials include luciferase, luciferin, and aequorin; and suitableradioactive nuclides include ¹²⁵I, ¹³¹I, ¹¹¹In, and ⁹⁹Tc.

In one embodiment, the invention also provides diagnostic kits,comprising an NKCC1 capture reagent, e.g. an antibody. In addition, sucha kit may optionally comprise one or more of the following:

-   -   (1) instructions for using the capture reagent for diagnosis,        prognosis, therapeutic monitoring or any combination of these        applications;    -   (2) a labelled binding partner to the capture reagent;    -   (3) a solid phase (such as a reagent strip) upon which the        capture reagent is immobilised; and    -   (4) a label or insert indicating regulatory approval for        diagnostic, prognostic or therapeutic use or any combination        thereof.

If no labelled binding partner to the capture reagent is provided, theanti-NKCC1 capture reagent itself can be labelled with a detectablemarker, e.g. a chemiluminescent, enzymatic, fluorescent, or radioactivemoiety.

NKCC1 antibodies can be conjugated to a therapeutic agent or drug moietyto modify a given biological response. The therapeutic agent or drugmoiety is not to be construed as limited to classical chemicaltherapeutic agents. For example, the drug moiety may be a protein orpolypeptide possessing a desired biological activity. Such proteins mayinclude, for example, a toxin such as abrin, ricin A, pseudomonasexotoxin, or diphtheria toxin; a protein such as tumour necrosis factor,α-interferon, β-interferon, nerve growth factor, platelet derived growthfactor, tissue plasminogen activator, a thrombotic agent or ananti-angiogenic agent, e.g. angiostatin or endostatin; or, a biologicalresponse modifier such as a lymphokine, interleukin-1 (IL-1),interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophagecolony stimulating factor (GM-CSF), granulocyte colony stimulatingfactor (G-CSF), nerve growth factor (NGF) or other growth factor. Othertherapeutic moieties may include radionuclides such as ¹¹¹In and ⁹⁰Y;antibiotics, e.g. calicheamicin; or drugs such as but not limited to,alkylphosphocholines, topoisomerase I inhibitors, taxoids and suramin.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g. Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982) and Dubowchik et al., 1999, Pharmacologyand Therapeutics, 83, 67-123.

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described in U.S. Pat. No.4,676,980.

An antibody with or without a therapeutic moiety conjugated to it can beused as a therapeutic that is administered alone or in combination withcytotoxic factor(s) and/or cytokine(s).

A further aspect of the invention provides methods of screening foractive agents that modulate (e.g. upregulate or downregulate) acharacteristic of, e.g. the expression or the enzymatic or bindingactivity of an NKCC1 polypeptide. The present invention also providesassays for use in drug discovery in order to identify or verify theefficacy of agents for treatment or prevention of breast, lung and/orpancreatic cancer. Candidate agents can be assayed for their ability tomodulate levels of a polypeptide as defined herein in a subject havingbreast, lung and/or pancreatic cancer. Agents able to modulate levels ofan NKCC1 polypeptide in a subject having breast, lung and/or pancreaticcancer towards levels found in subjects free from breast, lung and/orpancreatic cancer or to produce similar changes in experimental animalmodels of breast, lung and/or pancreatic cancer can be used as leadagents for further drug discovery, or used therapeutically. Expressionof an NKCC1 polypeptide can be assayed by, for example, immunoassays,gel electrophoresis followed by visualisation, detection of activity, orany other method taught herein or known to those skilled in the art.Such assays can be used to screen candidate agents, in clinicalmonitoring or in drug development, where abundance of an NKCC1polypeptide can serve as a surrogate marker for clinical disease.

Therefore, the present invention provides methods for identifying activeagents that bind to an NKCC1 polypeptide or have a modulatory effect(e.g. stimulatory, inhibitory, up-regulatory or down-regulatory) effecton the expression or activity of an NKCC1 polypeptide. Examples ofagents, include, but are not limited to, nucleic acids (e.g. DNA andRNA), carbohydrates, lipids, proteins, peptides, peptidomimetics,agonists, antagonists, small molecules and other drugs. Candidate agentscan be obtained using any of the numerous suitable approaches incombinatorial library methods known in the art, including: biologicallibraries; spatially addressable parallel solid phase or solution phaselibraries; synthetic library methods requiring deconvolution; the“one-bead one-compound” library method; and synthetic library methodsusing affinity chromatography selection. The biological library approachis limited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds (Ian, 1997, Anticancer Drug Des. 12:145; U.S. Pat. No.5,738,996; and U.S. Pat. No. 5,807,683).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al., 1993, Proc. Natl. Acad.Sci. USA 90:6909; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91:11422;Zuckermann et al., 1994, J. Med. Chem. 37:2678; Cho et al., 1993,Science 261:1303; Carrell et al., 1994, Angew. Chem. Int. Ed. Engl.33:2059; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2061; andGallop et al., 1994, J. Med. Chem. 37:1233.

Libraries of agents may be presented, e.g. in solution (e.g. Houghten,1992, Bio/Techniques 13:412-421), or on beads (Lam, 1991, Nature354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria (U.S. Pat.No. 5,223,409), spores (U.S. Pat. No. 5,571,698; U.S. Pat. No.5,403,484; and U.S. Pat. No. 5,223,409), plasmids (Cull et al., 1992,Proc. Natl. Acad. Sci. USA 89:1865-1869) or phage (Scott and Smith,1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla etal., 1990, Proc. Natl. Acad. Sci. USA 87:6378-6382; and Felici, 1991, J.Mol. Biol. 222:301-310).

In one embodiment, agents that interact with (i.e. bind to) an NKCC1polypeptide are identified in a cell-based assay system. In accordancewith this embodiment, cells expressing an NKCC1 polypeptide arecontacted with a candidate agent or a control agent and the ability ofthe candidate agent to interact with the polypeptide is determined. Ifdesired, this assay may be used to screen a plurality (e.g. a library)of candidate agents. The cell, for example, can be of prokaryotic origin(e.g. E. coli) or eukaryotic origin (e.g. yeast or mammalian). Further,the cells can express the NKCC1 polypeptide endogenously or begenetically engineered to express said polypeptide. In some embodiments,the NKCC1 polypeptide or the candidate agent is labelled, for examplewith a radioactive label (such as ³²P, ³⁵S or ¹²⁵I or a fluorescentlabel (such as fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde or fluorescamine) to enabledetection of an interaction between a polypeptide and a candidate agent.The ability of the candidate agent to interact directly or indirectlywith the NKCC1 polypeptide can be determined by methods known to thoseof skill in the art. For example, the interaction between a candidateagent and a polypeptide can be determined by flow cytometry, ascintillation assay, immunoprecipitation or western blot analysis.

In another embodiment, agents that interact with (i.e. bind to) an NKCC1polypeptide are identified in a cell-free assay system. In accordancewith this embodiment, a native or recombinant NKCC1 polypeptide iscontacted with a candidate agent or a control agent and the ability ofthe candidate agent to interact with the polypeptide is determined. Ifdesired, this assay may be used to screen a plurality (e.g. a library)of candidate agents. Preferably, the polypeptide is first immobilised,by, for example, contacting the polypeptide with an immobilised antibodywhich specifically recognises and binds it, or by contacting a purifiedpreparation of polypeptide with a surface designed to bind proteins. Thepolypeptide may be partially or completely purified (e.g. partially orcompletely free of other polypeptides) or part of a cell lysate.Further, the polypeptide may be a fusion protein comprising the NKCC1polypeptide or a biologically active portion thereof and a domain suchas glutathionine-S-transferase. Alternatively, the polypeptide can bebiotinylated using techniques well known to those of skill in the art(e.g. biotinylation kit, Pierce Chemicals; Rockford, Ill.). The abilityof the candidate agent to interact with the polypeptide can be can bedetermined by methods known to those of skill in the art.

In another embodiment, a cell-based assay system is used to identifyactive agents that bind to or modulate the activity of a protein, suchas an enzyme, or a biologically active portion thereof, which isresponsible for the production or degradation of the NKCC1 polypeptideor is responsible for the post-translational modification of thepolypeptide. In a primary screen, a plurality (e.g. a library) of agentsare contacted with cells that naturally or recombinantly express: (i) anNKCC1 polypeptide; and (ii) a protein that is responsible for processingof the NKCC1 polypeptide in order to identify compounds that modulatethe production, degradation, or post-translational modification of thepolypeptide. If desired, active agents identified in the primary screencan then be assayed in a secondary screen against cells naturally orrecombinantly expressing the specific polypeptide of interest. Theability of the candidate agent to modulate the production, degradationor post-translational modification of an NKCC1 polypeptide can bedetermined by methods known to those of skill in the art, includingwithout limitation, flow cytometry, a scintillation assay,immunoprecipitation and Western blot analysis.

In another embodiment, agents that competitively interact with (i.e.competitively bind to) an NKCC1 polypeptide are identified in acompetitive binding assay. In accordance with this embodiment, cellsexpressing the polypeptide are contacted with a candidate agent and anagent known to interact with the polypeptide; the ability of thecandidate agent to competitively interact with the polypeptide is thendetermined. Alternatively, agents that competitively interact with (i.e.competitively bind to) a polypeptide are identified in a cell-free assaysystem by contacting the polypeptide with a candidate agent and an agentknown to interact with the polypeptide. As stated above, the ability ofthe candidate agent to interact with a polypeptide for use in theinvention can be determined by methods known to those of skill in theart. These assays, whether cell-based or cell-free, can be used toscreen a plurality (e.g. a library) of candidate agents.

In another embodiment, active agents that modulate (i.e. upregulate ordownregulate) the expression of an NKCC1 polypeptide or an NKCC1 nucleicacid are identified by contacting cells (e.g. cells of prokaryoticorigin or eukaryotic origin) expressing the polypeptide or the nucleicacid with a candidate agent or a control agent (e.g. phosphate bufferedsaline (PBS)) and determining the expression of the polypeptide or thenucleic acid encoding the polypeptide. The level of expression of anNKCC1 polypeptide or NKCC1 nucleic acid in the presence of the candidateagent is compared to the level of expression of the polypeptide ornucleic acid in the absence of the candidate agent (e.g. in the presenceof a control agent). The candidate agent can then be identified as amodulator of the expression of the polypeptide based on this comparison.For example, when expression of the polypeptide or nucleic acid issignificantly greater in the presence of the candidate agent than in itsabsence, the candidate agent is identified as a stimulator of expressionof the polypeptide or nucleic acid. Alternatively, when expression ofthe polypeptide or nucleic acid is significantly less in the presence ofthe candidate agent than in its absence, the candidate agent isidentified as an inhibitor of the expression of the polypeptide ornucleic acid. The level of expression of an NKCC1 polypeptide, or thenucleic acid, can be determined by methods known to those of skill inthe art based on the present description. For example, DNA or mRNAexpression can be assessed by Northern blot analysis or RT-PCR, andprotein levels can be assessed by Western blot analysis.

In another embodiment, active agents that modulate the activity of anNKCC1 polypeptide are identified by contacting a preparation containingthe polypeptide, or cells (e.g. prokaryotic or eukaryotic cells)expressing the polypeptide with a candidate agent or a control agent anddetermining the ability of the candidate agent to modulate (e.g.stimulate or inhibit) the activity of the polypeptide. The activity ofan NKCC1 polypeptide can be assessed by detecting its effect on a“downstream effector” for example, but without limitation, induction ofa cellular signal transduction pathway of the polypeptide (e.g.intracellular Ca²⁺, diacylglycerol, IP₃, etc.), detecting catalytic orenzymatic activity of the target on a suitable substrate, detecting theinduction of a reporter gene (e.g. a regulatory element that isresponsive to an NKCC1 polypeptide and is operably linked to a nucleicacid encoding a detectable marker, e.g. luciferase), or detecting acellular response, for example, cellular differentiation, or cellproliferation as the case may be, based on the present description,techniques known to those of skill in the art can be used for measuringthese activities (see, e.g. U.S. Pat. No. 5,401,639). The candidateagent can then be identified as a modulator of the activity of an NKCC1polypeptide by comparing the effects of the candidate agent to thecontrol agent. Suitable control agents include phosphate buffered saline(PBS) and normal saline (NS).

In another embodiment, active agents that modulate the expression,activity or both the expression and activity of an NKCC1 polypeptide areidentified in an animal model. Examples of suitable animals include, butare not limited to, mice, rats, rabbits, monkeys, guinea pigs, dogs andcats. Preferably, the animal used represents a model of breast,pancreatic and/or lung cancer. In accordance with this embodiment, thecandidate agent or a control agent is administered (e.g. orally,rectally or parenterally such as intraperitoneally or intravenously) toa suitable animal and the effect on the expression, activity or bothexpression and activity of the polypeptide is determined. Changes in theexpression of a polypeptide can be assessed by any suitable methoddescribed above, based on the present description. Alternatively, agentsmay be identified by monitoring the effect of their administration onsymptoms associated with the disease or condition to be treated (e.g. toameliorate symptoms or to delay onset or slow the progression of thedisease). Therefore in a further embodiment, agents that reduce theseverity of one or more symptoms associated with the disease or thatslow the progression of the disease in a group of mammals treated withthe candidate agent, compared to a untreated group of mammals areidentified as potential active agents for the treatment of the disease.Techniques known to physicians familiar with breast, pancreatic and/orlung cancer can be used to determine whether a candidate agent hasaltered one or more symptoms associated with said diseases. For example,a candidate agent that reduces tumour burden in a subject having breast,pancreatic and/or lung cancer will be beneficial for treating breast,pancreatic and/or lung cancer patients.

In a particular embodiment, the methods of screening as described abovemay additionally comprise selecting an agent which modulates theexpression or activity of said polypeptide or the expression of saidnucleic acid molecule, for further testing or therapeutic orprophylactic use as an anti-breast, lung and/or pancreatic cancer agent.

In yet another embodiment, an NKCC1 polypeptide is used as a “baitprotein” in a two-hybrid assay or three hybrid assay to identify otherproteins that bind to or interact with the polypeptide (see, e.g. U.S.Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al.(1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993)Bio/Techniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696;and WO 94/10300). As those skilled in the art will appreciate, suchbinding proteins are also likely to be involved in the propagation ofsignals by the NKCC1 polypeptides as, for example, upstream ordownstream elements of a signalling pathway involving the NKCC1polypeptides.

This invention further provides NKCC1 polypeptides, NKCC1 nucleic acids,NKCC1 antibodies, agents that modulate the expression or activity of aNKCC1 polypeptide, that interact with a NKCC1 polypeptide or thatmodulate the expression of a NKCC1 nucleic acid, including thoseidentified by the above-described screening methods and uses thereof fortreatments as described herein. Hereinafter, the agents, NKCC1polypeptides, NKCC1 nucleic acids and NKCC1 antibodies are referred toas “active agents”. The term “treatment” includes either therapeutic orprophylactic therapy. When a reference is made herein to a method oftreating or preventing a disease or condition using a particular activeagent or combination of agents, it is to be understood that such areference is intended to include the use of that active agent orcombination of agents in the preparation of a medicament for thetreatment of said disease or condition.

The invention also provides for treatment and/or prevention of breast,pancreatic and/or lung cancer by administration of an active agent.

In a further aspect, the present invention provides the use of an NKCC1polypeptide in the production of a composition for the treatment orprophylaxis of breast, lung and/or pancreatic cancer, wherein thecomposition is a vaccine. The vaccine optionally comprises one or moresuitable adjuvants. Examples of adjuvants well-known in the art includeinorganic gels, such as aluminium hydroxide, and water-in-oil emulsions,such as incomplete Freund's adjuvant. Other useful adjuvants will bewell known to the skilled person.

In yet further aspects, the present invention provides:

-   -   (a) the use of an NKCC1 polypeptide in the preparation of an        immunogenic composition, preferably a vaccine;    -   (b) the use of such an immunogenic composition in inducing an        immune response in a subject; and    -   (c) a method for the treatment or prophylaxis of breast, lung        and/or pancreatic cancer in a subject, or of vaccinating a        subject against breast, lung and/or pancreatic cancer which        comprises the step of administering to the subject an effective        amount of an NKCC1 polypeptide, preferably as a vaccine.

In one embodiment, one or more active agents are administered alone orin combination e.g. simultaneously, sequentially or separately, with oneor more additional treatments or therapeutic agents for breast, lungand/or pancreatic cancer. Examples of such treatments include, surgeryand radiation therapy. Examples of therapeutic compounds include but arenot limited to cyclophosphamide (Cytoxan™); methotrexate(Methotrexate™); 5-fluorouracil (5-FU); paclitaxel (Taxol); docetaxel(Taxotere™); vincristine (Oncovin™); vinblastine (Velban™); vinorelbine(Navelbine™); doxorubicin (Adriamycin); tamoxifen (Nolvadex™);toremifene (Fareston™); megestrol acetate (Megace™); anastrozole(Arimidex™); goserelin (Zoladex™); anti-HER2 monoclonal antibody(Herceptin™); capecitabine (Xeloda™) and raloxifene hydrochloride(Evista™).

As discussed herein, active agents of the invention find use in thetreatment or prophylaxis of breast, pancreatic and/or lung cancer. Thus,in a further aspect, the present invention provides a pharmaceuticalcomposition comprising at least one active agent, optionally togetherwith one or more pharmaceutically acceptable excipients, carriers ordiluents. In one embodiment, the pharmaceutical composition is for useas a vaccine and so any additional components will be acceptable forvaccine use. In addition, the skilled person will appreciate that one ormore suitable adjuvants may be added to such vaccine preparations.

The composition will usually be supplied as part of a sterile,pharmaceutical composition that will normally include a pharmaceuticallyacceptable carrier. This pharmaceutical composition may be in anysuitable form (depending upon the desired method of administering it toa patient).

It may be provided in unit dosage form will generally be provided in asealed container and may be provided as part of a kit. Such a kit wouldnormally (although not necessarily) include instructions for use. It mayinclude a plurality of said unit dosage forms.

The pharmaceutical composition may be adapted for administration by anyappropriate route, for example by the oral (including buccal orsublingual), rectal, nasal, topical (including buccal, sublingual ortransdermal), vaginal or parenteral (including subcutaneous,intramuscular, intravenous or intradermal) route. Such compositions maybe prepared by any method known in the art of pharmacy, for example byadmixing the active ingredient with the carrier(s) or excipient(s) understerile conditions.

Pharmaceutical compositions adapted for oral administration may bepresented as discrete units such as capsules or tablets; as powders orgranules; as solutions, syrups or suspensions (in aqueous or non-aqueousliquids; or as edible foams or whips; or as emulsions).

Suitable excipients for tablets or hard gelatine capsules includelactose, maize starch or derivatives thereof, stearic acid or saltsthereof.

Suitable excipients for use with soft gelatine capsules include forexample vegetable oils, waxes, fats, semi-solid, or liquid polyols etc.

For the preparation of solutions and syrups, excipients which may beused include for example water, polyols and sugars. For the preparationof suspensions, oils (e.g. vegetable oils) may be used to provideoil-in-water or water in oil suspensions.

Pharmaceutical compositions adapted for transdermal administration maybe presented as discrete patches intended to remain in intimate contactwith the epidermis of the recipient for a prolonged period of time. Forexample, the active ingredient may be delivered from the patch byiontophoresis as generally described in Pharmaceutical Research,3(6):318 (1986).

Pharmaceutical compositions adapted for topical administration may beformulated as ointments, creams, suspensions, lotions, powders,solutions, pastes, gels, sprays, aerosols or oils. For infections of theeye or other external tissues, for example mouth and skin, thecompositions are preferably applied as a topical ointment or cream. Whenformulated in an ointment, the active agent may be employed with eithera paraffinic or a water-miscible ointment base. Alternatively, theactive agent may be formulated in a cream with an oil-in-water creambase or a water-in-oil base. Pharmaceutical compositions adapted fortopical administration to the eye include eye drops wherein the activeagent is dissolved or suspended in a suitable carrier, especially anaqueous solvent. Pharmaceutical compositions adapted for topicaladministration in the mouth include lozenges, pastilles and mouthwashes.

Pharmaceutical compositions adapted for rectal administration may bepresented as suppositories or enemas.

Pharmaceutical compositions adapted for nasal administration wherein thecarrier is a solid include a coarse powder having a particle size forexample in the range 20 to 500 microns which is administered by rapidinhalation through the nasal passage from a container of the powder heldclose up to the nose. Suitable compositions wherein the carrier is aliquid, for administration as a nasal spray or as nasal drops, includeaqueous or oil solutions of the active agent.

Pharmaceutical compositions adapted for administration by inhalationinclude fine particle dusts or mists which may be generated by means ofvarious types of metered dose pressurised aerosols, nebulisers orinsufflators.

Pharmaceutical compositions adapted for vaginal administration may bepresented as pessaries, tampons, creams, gels, pastes, foams or spraycompositions.

Pharmaceutical compositions adapted for parenteral administrationinclude aqueous and non-aqueous sterile injection solution which maycontain anti-oxidants, buffers, bacteriostats and solutes which renderthe composition substantially isotonic with the blood of the intendedrecipient; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents and thickening agents. Excipients which may beused for injectable solutions include water, alcohols, polyols,glycerine and vegetable oils, for example. The compositions may bepresented in unit-dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilised)condition requiring only the addition of the sterile liquid carried, forexample water for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions may be prepared from sterilepowders, granules and tablets.

The pharmaceutical compositions may contain preserving agents,solubilising agents, stabilising agents, wetting agents, emulsifiers,sweeteners, colourants, odourants, salts (polypeptides for use in thepresent invention may themselves be provided in the form of apharmaceutically acceptable salt), buffers, coating agents orantioxidants.

Dosages of the active agents for use in the present invention can varybetween wide limits, depending upon the stage of the breast, lung and/orpancreatic cancer, the age and condition of the individual to betreated, etc. and a physician will ultimately determine appropriatedosages to be used. This dosage may be repeated as often as appropriate.If side effects develop the amount and/or frequency of the dosage can bereduced, in accordance with normal clinical practice.

In view of the importance of NKCC1 in breast, pancreatic and/or lungcancer the following form additional aspects of the present invention:

-   -   i) a method for monitoring/assessing breast, pancreatic and/or        lung cancer treatment in a patient, which comprises the step of        determining the presence or absence and/or quantifying an NKCC1        polypeptide or an NKCC1 nucleic acid molecule in a biological        sample obtained from said patient.    -   ii) methods of treating breast, pancreatic and/or lung cancer,        comprising administering to a patient a therapeutically        effective amount of an active agent that interacts with or        modulates (e.g. upregulates or downregulates) or complements the        expression or the biological activity (or both) of an NKCC1        polypeptide in patients having breast, pancreatic and/or lung        cancer, in order to (a) prevent the onset or development of        breast, lung and/or pancreatic cancer; (b) prevent the        progression of breast, pancreatic and/or lung cancer; or (c)        ameliorate the symptoms of breast, pancreatic and/or lung        cancer.    -   iii) the use of an active agent, which interacts with, or        modulates the expression or activity of an NKCC1 polypeptide in        the preparation of a composition for the treatment of breast,        lung and/or pancreatic cancer.    -   iv) a method for the prophylaxis and/or treatment of breast,        lung and/or pancreatic cancer in a subject, which comprises        administering to said subject a therapeutically effective amount        of an antibody which binds to at least one NKCC1 polypeptide.    -   v) the use of an antibody which binds to at least one NKCC1        polypeptide in the preparation of a composition for use in the        prophylaxis and/or treatment of breast, lung and/or pancreatic        cancer.

Preferred features of each aspect of the invention are as for each ofthe other aspects mutatis mutandis.

The contents of each reference, patent and patent application cited inthis application is hereby incorporated by reference in its entirety.

The invention will now be described with reference to the followingexamples, which should not in any way be construed as limiting the scopeof the present invention. The examples refer to the figures in which:

FIG. 1: shows the nucleotide and amino acid sequences of NKCC1 (GenBankaccession: U30246; SwissProt accession: P55011). The tandem spectra usedto identify NKCC1 in breast and pancreatic cancer cell line membranepreparations are shown in bold, italicised, and underlined. Massesassigned to NKCC1 are shown in bold and italicised (see below).

FIG. 2: shows the normal tissue distribution of NKCC1 mRNA. Levels ofmRNA in normal tissues were quantified by real time RT-PCR. mRNA levelsare expressed as the number of copies ng⁻¹ cDNA.

FIG. 3: shows the expression of NKCC1 mRNA in normal and breast cancertissues. Levels of NKCC1 mRNA in donor matched normal and adjacenttumour tissues were measured by real time RT-PCR. mRNA levels areexpressed as the number of copies ng⁻¹ cDNA.

FIG. 4: shows the expression of NKCC1 mRNA in normal pancreatic tissues,pancreatic cancer tissues, and normal and tumour-derived pancreatic celllines. Levels of NKCC1 mRNA were measured by real time RT-PCR. mRNAlevels are expressed as the number of copies ng⁻¹ cDNA.

EXAMPLE 1 Identification of NKCC1

NKCC1 was Isolated from Breast Cancer (MDA MB 468/BT474) and Pancreatic(HPAFII) Cell Lines.

Cells were grown to confluence in 15 cm² cell culture dishes beforefractionation. Before harvest and extraction, the cells (approx. 2×10⁹cells) were washed three times with PBS-CM. Cells were scraped fromculture dishes in ice-cold PBS-CM (5 ml per 2×10⁸ cells) using a plasticcell lifter. The cells were then centrifuged at 1000×g for 5 min at +4°C. The supernatant was removed and the cells were resuspended in 10 mlof homogenisation buffer (250 mM Sucrose in 10 mM HEPES, 1 mM EDTA 1 nMVanadate, 0.02% Azide) followed by centrifugation at 1000×g for 5 min at+4° C. The supernatant was removed. The cell pellet was then resuspendedin 5× packed cell volume with homogenisation buffer plus proteaseinhibitors (Sigma). A ball bearing homogeniser (BBH) (8.002 mm ball) waschilled and rinsed with homogenisation buffer. The cell suspension wastaken up in a 2 ml syringe and attached to one side of the BBH. Anothersyringe was attached to the other side of the BBH. The cell mixture wasfed through the chamber up to five times. Breakage of the cells wasmonitored using a microscope and when the cells were sufficiently lysedthe resulting mixture was centrifuged at 1000×g for 5 min at +4° C.

The resulting supernatant (PNS) was retained and 1 ml of homogenisationbuffer was added to the nuclear pellet followed by centrifugation at1000×g for 5 min. The latter two fractions were pooled and centrifugedat 3000×g for 10 min at +4° C. The 3000×g supernatant was layered onto a2 ml 60% sucrose cushion in SW40 or SW60 tube and centrifuged at 100000×g for 45 min with slow acceleration and deceleration. The crudeplasma membrane was evident as a discrete layer on top of the sucrosecushion. The upper layer was removed (cytosol) and the plasma membranewas collected using a pasteur pipette. The % sucrose of crude plasmamembrane fraction was determined using a refractometer. The membranepreparation was diluted with HEPES buffer to reduce the sucrose contentto below 15%. The crude plasma membrane preparation was layered onpreformed 15 to 60% sucrose gradient in SW40 tube and spun at 100 000×gfor 17 h with slow acceleration and deceleration.

The sucrose gradient was fractionated using the gradient unloader (speed0.5, distance 2.5, fractions 35). The protein content of the fractionswas measured and 10 micrograms of protein was run on a 4-20% acrylamide1D gel (Novex) and subject to western blotting with antibodies toTransferrin Receptor, Oxidoreductase II and Calnexin.

Plasma membrane fractions that had transferrin immunoreactivity but nooxidoreductase II or calnexin immunoreactivity were identified. Thesesucrose fractions were pooled and diluted at least four times with 10 mMHEPES, 1 mM EDTA 1 mM Vanadate, 0.02% Azide. The diluted sucrosefraction was added to a SW40 or SW60 tube and centrifuged at 100 000×gfor 45 min with slow acceleration and deceleration. The supernatant wasremoved from the membrane pellet and the pellet washed three times withPBS-CM. The membrane pellet was solubulized in 2% SDS in 63 mM TrisHCl,pH 7.4. A protein assay was performed followed by the addition ofmercaptoethanol (2% final), glycerol (10%) and bromopheneol blue(0.0025% final). The extracted protein sample was finally solubilized in1D lysis buffer to a final protein concentration of microgram/microlitreand the proteins separated by 1D PAGE.

Mass Spectrometry

Proteins excised from the 1D gel were digested with trypsin and analysedby MALDI-TOF-MS (Voyager STR, Applied Biosystems) using a 337 nmwavelength laser for desorption and the reflectron mode of analysis.Selected masses for NKCC1 were further characterised by tandem massspectrometry using a QTOF-MS equipped with a nanospray ion source,(Micromass UK Ltd.). Prior to MALDI analysis the samples were desaltedand concentrated using C18 Zip Tips™ (Millipore). Samples for tandemmass spectrometry (MS) were purified using a nano LC system (LCPackings, Amsterdam, The Netherlands) incorporating C18 SPE material.

Using the SEQUEST search program (Eng et al., 1994, J. Am. Soc. MassSpectrom. 5:976 989), uninterpreted tandem mass spectra of trypticdigest peptides were searched against a database of public domainproteins constructed of protein entries in the non-redundant databaseheld by the National Centre for Biotechnology Information (NCBI). Thisdatabase is accessible at http://www.ncbi.nlm.nih.gov/ and alsoconstructed of Expressed Sequence Tags entries(http://www.ncbi.nlm.nih.gov/dbEST/index.html). As a result of sequencedatabase searching tandem amino acid sequences were found to match aSwissProt accession number: P55011 (Bumetanide-sensitivesodium-(potassium)-chloride cotransporter 1), see FIG. 1 (SEQ ID NO: 1),matched tandem sequences are shown in bold.

Additionally, sequences were identified using peptide mass data derivedfrom mass spectrometer analysis and the MOWSE database search procedure.Peptide mass information can provide a ‘fingerprint’ signaturesufficiently discriminating to allow for the unique and rapididentification of unknown sample proteins, independent of otheranalytical methods such as protein sequence analysis. Practicalexperience has shown that sample proteins can be uniquely identifiedusing as few as 3-4 experimentally determined peptide masses whenscreened against a fragment database derived from over 50,000 proteins(D. J. C. Pappin, P. Hojrup and A. J. Bleasby ‘Rapid Identification ofProteins by Peptide-Mass Fingerprinting’. Current Biology (1993), vol 3,327-332. and http://www.hgmp.mrc.ac.uk/Bioinformatics/Webapp/mowse/).The version of the code used for a MOWSE database search had thefollowing modifications: the size of the parent protein is not includedin the calculation such that large proteins such as titin no longer biasthe score; instead the theoretical frequency of a peptide of mass (x) isestimated using the mass (x) of the peptide and the mean mass of anamino acid whilst allowing for a probability of 0.2 for a missedinternal cleavage (by trypsin) and 0.1 for the probability of theoccurrence of a proteolytic cleavage site.

The score assigned to a match on a peptide mass (x) is the logarithm ofthe probability of finding such a match at random and is inverselyproportional to the frequency of fragments of that mass.

The score is thus calculated using the linear regression formula foundby plotting the score for a match on peptide mass (x) against the mass(x). Two mass matches to predicted trypsin fragments were identified inthis manner for NKCC1, which also matched to P55011, see FIG. 1 (SEQ IDNO: 1), the sequences which were identified through mass matching areshown in italics and underlined. The accuracy of this method was 20 ppm.

EXAMPLE 2 Expression of NKCC1 mRNA in Human Tissues

Real time quantitative RT-PCR was used (Heid, C. A., et al., Genome Res.6, 986-994 (1996); Morrison, T. B., et al., Biotechniques 24, 954-958(1998)) to analyse the distribution of NKCC1 mRNA in normal humantissues (FIG. 2), donor matched tumour and adjacent normal tissues fromseven breast cancer patients, (FIG. 3), normal and tumour-derivedpancreatic cell lines and tissue specimens (FIG. 4).

Quantification of NKCC1 mRNA by RT-PCR

Real-time RT-PCR was used to quantitatively measure NKCC1 expression innormal human tissue mRNAs (Clontech), donor matched tumour and adjacentnormal tissues from breast cancer patients, normal and tumour-derivedpancreatic cell lines and tissue specimens. Ethical approval for thenormal and tumour tissue samples was obtained at surgery (University ofOxford, UK). The primers used for PCR were as follows: (SEQ ID NO: 3)sense, 5′ cacctactacctgcgcaccttc 3′, (SEQ ID NO: 4) antisense, 5′gaccacagcatctctggttgga 3′,

Reactions contained 5 ng cDNA (prepared using Superscript first strandsynthesis for RT-PCR kit (Life Technologies)), SYBR green sequencedetection reagents (PE Biosystems), sense and antisense primers, andwere assayed on an ABI7700 sequence detection system (PE Biosystems).The PCR conditions were 1 cycle at 50° C. for 2 min, 1 cycle at 95° C.for 10 min, and 40 cycles of 95° C. for 15 sec, 65° C. for 1 min. Theaccumulation of PCR product was measured in real time as the increase inSYBR green fluorescence, and the data were analysed using the SequenceDetector program v1.6.3 (PE Biosystems). Standard curves relatinginitial template copy number to fluorescence and amplification cyclewere generated using the amplified PCR product as a template, and wereused to calculate NKCC1 copy number in each sample.

Overall the distribution of NKCC1 mRNA was low in normal tissues, withthe highest levels of mRNA expression seen in mammary, prostate, testisand brain tissues (FIG. 2).

The expression of NKCC1 mRNA in clinical breast carcinoma tissues wascompared with the matched adjacent normal tissue from 7 breast cancerpatients (FIG. 3). NKCC1 expression was increased in all of the tumoursamples, relative to their matched control tissue, with six of the sevensamples showing a greater than 6-fold increase in expression.

NKCC1 mRNA expression was also analysed in normal and tumour pancreatictissues and cell lines (FIG. 4). NKCC1 expression was found to besignificantly higher in the pancreatic tumour tissue (mean=69 copiesng⁻¹ cDNA) and tumour-derived cell lines (mean=1013 copies ng⁻¹, cDNA)than in the corresponding normal pancreatic tissues and cell lines(mean=16 and 5 copies per ng⁻¹ cDNA respectively).

EXAMPLE 3 Immunohistochemical Analysis of NKCC1 in Breast, Lung andPancreatic Cancer Tissues

To further illustrate the involvement of NKCC1 in breast cancer,immunohistochemistry with a specific anti-NKCC1 antibody was used toinvestigate NKCC1 protein expression in sections of multiple donorbreast ductal carcinoma tissues and in lung adenocarcinoma andpancreatic cancer tissues.

Antibody Generation.

The anti-NKCC1 polyclonal antibody was raised in guinea pigs immunisedwith 2 specific peptides (Immune Systems Limited, UK). Peptide sequenceswere chosen for synthesis based on plots of hydrophobicity,antigenicity, surface probability, and weak homology to other knownprotein family members. Peptides were synthesised using Fmoc chemistrywith a cysteine residue added to the end of each to enable specificthiol-reactive coupling of Keyhole Limpet Haemocyanin prior toimmunisation. The peptides used were; SKKPKGFFGYKC (SEQ ID NO: 5) andSGESEPAKGSEEAKGC (SEQ ID NO: 6). The antibodies were affinity purifiedusing columns of the above immobilised peptides.

NKCC1 Immunohistochemistry in Breast Carcinoma Tissue

Immunohistochemical analysis was carried out on formalin-fixedparaffin-embedded tissue microarrays containing mm sections of breastcarcinoma tissue from 55 donors as well as 20 sections of various normaltissues (Clinomics Laboratories Inc., 165 Tor Court, Pittsfield, Mass.01201). Slides were deparafinised by 2×5 min washes in xylene thenrehydrated through successive graded ethanol solutions and washed for 5min in PBS. Antigen retrieval was achieved by immersing the slides in0.01M citrate buffer (pH 6) and microwaving for 10 min at full power(950 W). In addition, detection with the antibody was improved byprotease treatment of the tissue with Autozyme (AbCam) for 10 min atroom temperature. The tissue was blocked in 10% donkey serum/PBS for 1 hbefore addition of 1.5 μg/ml primary polyclonal antibody (in 2.5% donkeyserum/PBS). Following 3 washes in PBS the tissue sections were incubatedwith biotin-conjugated secondary antibodies (Biotin-SP-conjugatedAffiniPure Donkey anti-guinea pig, Jackson ImmunoResearch) diluted at1:200 (2.5 μg/ml in 2.5% donkey serum/PBS) for 1 h. Slides were washed 3times in PBS and the tissue incubated with Streptavidin-HRP (JacksonImmunoResearch) diluted 1:100 (5 μg/ml in 2.5% donkey serum/PBS),followed by 3×5 min washes in PBS. Antibody signal was detected usingDAB substrate solution (Dako Ltd.) according to the manufacturers'instructions. An adjacent tissue array was counterstained forhematoxylin and eosin (Dako Ltd.) and images were captured by a digitalcamera attached to a light microscope.

NKCC1 immunostaining was seen in breast cancer tissue and it was clearlyapparent that NKCC1 is specifically and highly expressed in the ductalcarcinoma cells of the breast cancer tissue (compare with adjacentbreast tissue). In total 55 breast cancer donor tissues were examinedfor NKCC1 immunoreactivity. Of these 7 demonstrated very high NKCC1staining, 30 exhibited high/moderate staining, 13 exhibited weakstaining, and 5 showed no staining in the carcinoma cells.

NKCC1 Immunohistochemistry in Lung and Pancreatic Cancer Tissues

Immunohistochemical analysis was carried out on formalin-fixed paraffinembedded tissue microarrays containing 1 mm sections of carcinomatissues from a wide range of donors (obtained from ClinomicsLaboratories Inc., 165 Tor Court, Pittsfield, Mass. 01201). Slides weredeparafinized by 2×5 min washes in xylene then rehydrated throughsuccessive graded ethanol solutions and washed for 5 min in PBS. Antigenretrieval was achieved by immersing the slides in 0.01M citrate buffer(pH 6) and microwaving for 10 min at full power (950 W) then treatingthe tissue with pepsin (1 mg/ml) for 1 min at room temperature at pH 2.The tissue was blocked in 10% donkey serum/PBS for 1 h before additionof 1 μg/ml primary anti-NKCC1 polyclonal antibody (in 2.5% donkey serum)for 1 h. Following three washes in PBS the tissue sections wereincubated with biotin-conjugated secondary antibodies(Biotin-SP-conjugated AffiniPure Donkey anti-guinea pig IgG, JacksonImmunoResearch) diluted at 1:200 (2.5 μg/ml in 2.5% donkey serum/PBS)for 1 h. Slides were washed 3 times in PBS and the tissue incubated withStreptavidin-HRP (Jackson ImmunoResearch) diluted 1:100 (5 μg/ml in 2.5%donkey serum/PBS) for 1 h, followed by 3×5 min washes in PBS. Antibodysignal was detected using DAB substrate solution (Dako Ltd.) accordingto the manufacturer's instructions.

Increased staining of the sections for NKCC1 was seen in both lung andpancreatic tissue sections compared to adjacent control sections.

EXAMPLE 4 Cellular Localisation of NKCC1 in Recombinant Cell Lines

Fluorescent immunocytochemistry was used to assess the cellularlocalisation of NKCC1 in recombinant cell lines.

The full-length open reading frame of human NKCC1 (SEQ ID NO: 2) was PCRcloned into the plasmid vector pcDNA3.1 (Invitrogen). HEK293 cells andCH0-K1 cells were stably transfected with NKCC1.pcDNA3.1 plasmid(Invitrogen) using transfection reagent GeneJuice™ (Novagen) accordingto the manufacturer's instructions. A pool of HEK293 cells expressingfull-length NKCC1 was selected for growth in antibiotic-containingmedium (0.2 mg/ml G418). CHO-K1 cells expressing full-length NKCC1 weredilution cloned and selected for growth in antibiotic-containing medium(0.2 mg/ml G418). Two CHO-K1 clones, 4D8 and 3C5, were selected forfurther assessment.

Transfected and non-transfected HEK293 cells and CHO-K1 cells wereseeded onto 8-well chamber slides (Nalge Nunc) at a density of 6×10⁴cells per well. After 24 h of incubation at 37° C. and 5% CO₂ the mediawas removed from the slides and the plastic housing of the chambers wasalso removed. The slides were washed once in PBS in a Coplin jar forfive minutes. Excess PBS was removed from the slides and then they wereplaced in 100% acetone for 5 min. Following this incubation the slideswere air dried briefly and then laid flat in a humidifier chamber andincubated for 1 h with 500 μl of primary antibody appropriately dilutedin PBS 1% (w/v) BSA (2 μg/ml of guinea-pig anti-NKCC1 polyclonalantibody raised against peptide SEQ. ID NO: 5 (Immune Systems Limited,UK) or 2 μg/ml guinea-pig gamma globulin (Sigma)). Slides were thenwashed three times for 5 min per wash in PBS. The slides were-incubatedfor 1 h in a humidifier chamber with 500 μl of biotin-SP-conjugatedAffiniPure donkey anti-guinea pig antibody, (Jackson ImmunoResearch)diluted 1:200 in PBS 1% (w/v) BSA. Following this incubation the slideswere washed three times for 5 min per wash in PBS and then incubated for1 h in a humidifier chamber with 500 μl of Cy3-conjugated-Extravidin(Sigma) diluted 1:700 in PBS 1% (w/v) BSA. Following this incubation theslides were washed four times for 5 min per wash in PBS. Slides werethen incubated for 30 sec with 2 μg/nm BisBenzimide (Sigma) and thenwashed for 2 min in PBS. Slides were mounted with a coverslip influorescent enhancing media (Dako Ltd.) and then images were captured bydigital camera DC300F attached to a DMIRE2 fluorescence microscope(Leica Microsystems (UK) Ltd.)

The resulting images clearly showed that NKCC1 was highly expressed onthe plasma membrane of NKCC1-transfected HEK293 and CHO cells. Thisstaining is highly specific to the NKCC1 transfected cells sincestaining of non-transfected HEK293 and CHO-K1 cells with anti-NKCC1polyclonal antibody revealed very low levels of fluorescence. Controlguinea-pig IgG staining of NKCC1 transfected cells gave a very low levelof background fluorescence.

Thus, NKCC1 shows a restricted pattern of expression in normal humantissues, and is elevated in breast cancer tissues, lung cancer tissues,pancreatic cancer tissues and cancer cell lines. Immunocytochemistry incell lines transfected with NKCC1 demonstrated a clear membraneassociation, indicating that NKCC1 has potential in an immunotherapeuticbased approach to the treatment and/or prophylaxis of breast, lungand/or pancreatic cancer.

The present invention is not to be limited in terms of the particularembodiments described in this application, which are intended as singleillustrations of individual aspects of the invention. Functionallyequivalent methods and apparatus within the scope of the invention, inaddition to those enumerated herein, will be apparent to those skilledin the art from the foregoing description and accompanying drawings.Such modifications and variations are intended to fall within the scopeof the appended claims.

1. A method of screening for and/or diagnosis of breast, lung and/orpancreatic cancer in a subject and/or monitoring the effectiveness ofbreast, lung and/or pancreatic cancer therapy, which method comprisesthe step of detecting and/or quantifying in a biological sample obtainedfrom said subject: (i) an NKCC1 polypeptide which: a) comprises orconsists of the amino acid sequence shown in FIG. 1 (SEQ ID NO: 1); b)is a derivative having one or more amino acid substitutions,modifications, deletions or insertions relative to the amino acidsequence shown in FIG. 1 (SEQ ID NO: 1) which retains the immunologicalor biological activity of NKCC1; or c) is a fragment of a polypeptidehaving the sequence shown in FIG. 1 (SEQ ID NO: 1), which is at leastten amino acids long and has at least 70% sequence identity over thelength of the fragment; and/or (ii) a nucleic acid molecule which: d)comprises or consists of the DNA sequence shown in FIG. 1 (SEQ ID NO:2)or its RNA equivalent; e) is a sequence which codes for a polypeptide asdefined in a), b) or c); f) is a sequence which is complementary to thesequences of d) or e); g) is a sequence which codes for the samepolypeptide, as the sequences of d) or e); h) is a sequence which showssubstantial identity with any of those of d), e), f) or g), or i) is afragment of d), e), f), g) or h), which is at least 8 nucleotides inlength.
 2. The method of claim 1, wherein the level of said polypeptideor said nucleic acid is compared to a previously determined referencerange or control.
 3. The method according to claim 1, wherein the stepof detecting comprises: (a) contacting the sample with a capture reagentthat is specific for a polypeptide as defined in claim 1(i); and (b)detecting whether binding has occurred between the capture reagent andsaid polypeptide in the sample.
 4. The method according to claim 3,wherein step (b) comprises detecting the captured polypeptide using adirectly or indirectly labelled detection reagent.
 5. The methodaccording to claim 3, wherein the capture reagent is immobilised on asolid phase.
 6. An antibody, functionally-active fragment, derivative oranalogue thereof, that specifically binds to a polypeptide as defined inclaim 1(i).
 7. The method according to claim 1, wherein the NKCC1polypeptide is detected and/or quantified using an antibody thatspecifically binds to the polypeptide.
 8. An antibody according to claim6, wherein the antibody is monoclonal, polyclonal, chimeric, bispecific,humanised or is conjugated to a detectable substance, therapeuticmoiety, a second antibody or a fragment thereof, a cytotoxic agent or acytokine.
 9. A diagnostic kit comprising a capture reagent specific foran NKCC1 polypeptide as defined in claim 1(i), reagents and instructionsfor use.
 10. A method for the prophylaxis and/or treatment of breast,lung and/or pancreatic cancer in a subject, which comprisesadministering to said subject a therapeutically effective amount of: i)a polypeptide as defined in claim 1(i), or ii) a nucleic acid moleculeas defined in claim 1(ii).
 11. A method for the prophylaxis and/ortreatment of breast, lung and/or pancreatic cancer in a subject, whichcomprises administering to said subject a therapeutically effectiveamount of an antibody as defined in claim
 6. 12. (canceled) 13.(canceled)
 14. (canceled)
 15. (canceled)
 16. The method of claim 10,wherein the polypeptide and the nucleic acid molecule are prepared as acomposition, and wherein the composition is a vaccine.
 17. The method ofclaim 10, where the nucleic acid inhibits the expression of thepolypeptide as defined in claim 1(i).
 18. A method of screening foragents that interact with a polypeptide as defined in claim 1(i), saidmethod comprising: (a) contacting said polypeptide with a candidateagent; and (b) determining whether or not the candidate agent interactswith said polypeptide.
 19. The method according to claim 18, wherein thedetermination of interaction between the candidate agent and thepolypeptide comprises quantitatively detecting binding of the candidateagent and said polypeptide.
 20. A method of screening for anti-breast,pancreatic and/or lung cancer agents that modulate i) the expression oractivity of a polypeptide as defined in claim 1(i), or ii) theexpression of a nucleic acid molecule as defined in claim 1(ii), saidmethod comprising: a) comparing the expression or activity of saidpolypeptide or the expression of said nucleic acid molecule, in thepresence of a candidate agent with the expression or activity of saidpolypeptide or the expression of said nucleic acid molecule, in theabsence of the candidate agent or in the presence of a control agent;and b) determining whether the candidate agent causes the expression oractivity of said polypeptide or the expression of said nucleic acidmolecule, to change.
 21. The method of claim 20 wherein the expressionor activity level of said polypeptide or the expression level of saidnucleic acid molecule is compared with a predetermined reference range.22. The method of claims 20 wherein step b) additionally comprisesselecting an agent which modulates the expression or activity of saidpolypeptide or the expression of said nucleic acid molecule, for furthertesting or therapeutic or prophylactic use as an anti-breast, lungand/or pancreatic cancer agent.
 23. An active agent identified by themethod of claim 18 which interacts with said polypeptide or causes theexpression or activity of said polypeptide or the expression of saidnucleic acid molecule, to change.
 24. (canceled)
 25. An active agentwhich interacts with a polypeptide as defined in claim 1(i), whichmodulates the expression or activity of said polypeptide or whichmodulates the expression of a nucleic acid as defined in claim 1(ii),for use in the prophylaxis and/or treatment of breast, lung and/orpancreatic cancer.
 26. A method of prophylaxis and/or treatment ofbreast, lung and/or pancreatic cancer, which comprises administering tosaid subject a therapeutically effective amount of an active agent whichinteracts with a polypeptide as defined in claim 1(i), which modulatesthe expression or activity of said polypeptide or which modulates theexpression of a nucleic acid as defined in claim 1(ii).
 27. An activeagent identified by the method of claim 22 which interacts with saidpolypeptide or causes the expression or activity of said polypeptide orthe expression of said nucleic acid molecule, to change.